Duplexed parvovirus vectors

ABSTRACT

The present invention provides duplexed parvovirus vector genomes that are capable under appropriate conditions of forming a double-stranded molecule by intrastrand base-pairing. Also provided are duplexed parvovirus particles comprising the vector genome. Further disclosed are templates and methods for producing the duplexed vector genomes and duplexed parvovirus particles of the invention. Methods of administering these reagents to a cell or subject are also described. Preferably, the parvovirus capsid is an AAV capsid. It is further preferred that the vector genome comprises AAV terminal repeat sequences.

RELATED APPLICATION INFORMATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/208,604, filed Jun. 1, 2000, which is incorporated byreference herein in its entirety.

STATEMENT OF FEDERAL SUPPORT

[0002] The present invention was made, in part, with the support ofgrant numbers HL51818, HL 48347, and DK 54419 from the NationalInstitutes of Health. The United States government has certain rights tothis invention.

FIELD OF THE INVENTION

[0003] The present invention relates to reagents for gene delivery. Moreparticularly, the present invention relates to improved parvovirus-basedgene delivery vectors.

BACKGROUND OF THE INVENTION

[0004] Adeno-associated virus (AAV) is a nonpathogenic, helper dependentmember of the parvovirus family. One of the identifying characteristicsof this group is the encapsidation of a single-stranded DNA (ssDNA)genome. In the case of AAV, the separate plus or minus polarity strandsare packaged with equal frequency, and either is infectious. At each endof the ssDNA genome, a palindromic terminal repeat (TR) structurebase-pairs upon itself into a hairpin configuration. This serves as aprimer for cellular DNA polymerase to synthesize the complementarystrand after uncoating in the host cell. Adeno-associated virusgenerally requires a helper virus for a productive infection. Althoughadenovirus (Ad) usually serves this purpose, treatment of AAV infectedcells with UV irradiation or hydroxyurea (HU) will also allow limitedreplication.

[0005] Recombinant AAV (rAAV) gene delivery vectors also package ssDNAof plus or minus polarity, and must rely on cellular replication factorsfor synthesis of the complementary strand. While it was initiallyexpected that this step would be carried out spontaneously, by cellularDNA replication or repair pathways, this does not appear to be the case.Early work with rAAV vectors revealed that the ability to score markergene expression was dramatically enhanced when cells were co-infectedwith adenovirus, or transiently pretreated with genotoxic agents. Thisenhancement correlated with the formation of duplex DNA from thesingle-stranded virion DNA (vDNA). Similar induction of rAAV vectors hasbeen observed in vivo following treatment with Ad, ionizing radiation,or topoisomerase inhibitors. However, the effect was highly variablebetween different tissues and cell types. It has more recently beensuggested that reannealing of complementary vDNA from separate infectingrAAV particles may be an important pathway for rAAV transduction.

[0006] The requirement for complementary-strand synthesis, orrecruitment, is now considered to be a limiting factor in the efficiencyof rAAV vectors. The transduction rate for rAAV in mouse liver has beenestimated at approximately 5% of hepatocytes after portal vein infusionof 4.2×10¹⁰ particles. Subsequent experiments revealed that the rAAVvDNA had been taken up into the nuclei of virtually all of the liverhepatocytes, and that the transduction potential of these genomes couldbe rescued by co-infection with adenovirus. This is consistent with anearlier report of up to 25% of mouse hepatocytes transduced by 10¹⁰particles of rAAV in the presence of co-infecting adenovirus. Expressionfrom rAAV in liver tissue coincides with the formation of duplex DNA andthe vDNA appears to be lost if not converted to duplex within 5-13weeks. Further experiments suggest that a subpopulation of mousehepatocytes is transiently permissive for rAAV transduction in vivo.

[0007] Accordingly, the present invention addresses a need in the artfor improved parvovirus gene delivery vectors. In particular the presentinvention addresses the requirement for complementary strand synthesisby conventional AAV gene delivery vectors.

SUMMARY OF THE INVENTION

[0008] The single-stranded nature of the AAV genome may impact theexpression of rAAV vectors more than any other biological feature.Rather than rely on potentially variable cellular mechanisms to providea complementary-strand for rAAV vectors, it has now been found that thisproblem may be circumvented by packaging both strands as a single DNAmolecule. In the studies described herein, an increased efficiency oftransduction from duplexed vectors over conventional rAAV was observedin HeLa cells (5-140 fold). More importantly, unlike conventionalsingle-stranded AAV vectors, inhibitors of DNA replication did notaffect transduction from the duplexed vectors of the invention. Inaddition, the inventive duplexed parvovirus vectors displayed a morerapid onset and a higher level of transgene expression than did rAAVvectors in mouse hepatocytes in vivo. All of these biological attributessupport the generation and characterization of a new class of parvovirusvectors (delivering duplex DNA) that significantly contribute to theongoing development of parvovirus-based gene delivery systems.

[0009] Overall, a novel type of parvovirus vector that carries aduplexed genome, which results in co-packaging strands of plus and minuspolarity tethered together in a single molecule, has been constructedand characterized by the investigations described herein. Accordingly,the present invention provides a parvovirus particle comprising aparvovirus capsid (e.g., an AAV capsid) and a vector genome encoding aheterologous nucleotide sequence, where the vector genome isself-complementary, i.e., the vector genome is a dimeric invertedrepeat. The vector genome is preferably approximately the size of thewild-type parvovirus genome (e.g., the AAV genome) corresponding to theparvovirus capsid into which it will be packaged and comprises anappropriate packaging signal. The present invention further provides thevector genome described above and templates that encode the same.

[0010] As a further aspect, the present invention provides a duplexedparvovirus particle comprising: a parvovirus capsid and a vector genomecomprising in the 5′ to 3′ direction: (i) a 5′ parvovirus terminalrepeat sequence; (ii) a first heterologous nucleotide sequence; (iii) anon-resolvable parvovirus terminal repeat sequence; (iv) a separateheterologous nucleotide sequence that is essentially completelycomplementary to the first heterologous nucleotide sequence; and (v) a3′ parvovirus terminal repeat sequence; wherein the vector genome iscapable under appropriate conditions of intrastrand base-pairing betweenthe heterologous nucleotide sequences upon release from the parvoviruscapsid. A double-stranded sequence is formed by the base-pairing betweenthe complementary heterologous nucleotide sequences, which is a suitablesubstrate for gene expression (i.e., transcription and, optionally,translation) or a substrate for host recombination (i.e., a dsDNAtemplate) in a host cell without the need for host cell machinery toconvert the vector genome into a double-stranded form.

[0011] The designations of 5′ and 3′ with respect to the vector genome(or templates for producing the same, see below) does not indicate anyparticular direction of transcription from the double-stranded sequenceformed between the two complementary heterologous sequences. The “codingstrand” may be on either the 5′ or 3′ half of the virion DNA. Thoseskilled in the art will appreciate that the term “coding strand” isbeing used in its broadest sense to indicate the strand encoding thedesired transcript, and encompasses non-translated sequences as well,including antisense sequences. Thus, transcription may be initiated fromthe 5′ end of the first heterologous nucleotide sequence in the 5′ halfof the vector genome, or from the 5′ end of the complementaryheterologous nucleotide sequence on the 3′ half of the vector genome.

[0012] Alternatively stated, in the double-stranded vDNA formed byintrastrand base-pairing, transcription may be initiated from the openend or from the closed end (i.e., from the end closest to thenon-resolvable TR) of the hairpin structure.

[0013] According to this embodiment, the parvovirus capsid is preferablyan AAV capsid. It is further preferred that the parvovirus terminalrepeat sequences and/or the non-resolvable terminal repeat sequences areAAV sequences.

[0014] In particular embodiments, the duplexed parvovirus particlecomprises sufficient expression control sequences (e.g., a promoter) forexpression of the double-stranded sequence formed by intrastrandbase-pairing in the self-complementary vDNA.

[0015] The vector genome may further express two or more transcriptsfrom the double-stranded sequence formed by intrastrand base-pairing.

[0016] As a further aspect, the present invention provides a nucleotidesequence comprising a template for producing a virion DNA, the templatecomprising a heterologous nucleotide sequence flanked by a parvovirusterminal repeat sequence and a non-resolvable parvovirus terminal repeatsequence.

[0017] As a still further aspect, the present invention provides anucleotide sequence comprising a dimeric template for producing a virionDNA, the template comprising in the 5′ to 3′ direction: a 5′ parvovirusterminal repeat sequence; a first heterologous nucleotide sequence; anon-resolvable parvovirus terminal repeat sequence; a separateheterologous nucleotide sequence that is essentially completelycomplementary to the first heterologous nucleotide sequence; and a 3′parvovirus terminal repeat sequence; wherein the virion DNA is capableunder appropriate conditions of intrastrand base-pairing to form a dsDNAbetween the heterologous nucleotide sequences upon release from theparvovirus capsid.

[0018] Preferably, the parvovirus terminal repeat sequences and/orparvovirus non-resolvable terminal repeat sequences are AAV sequences.

[0019] The present invention further provides methods of producing andadministering the inventive duplexed parvovirus vectors of theinvention. In one particular embodiment, the present invention providesa method of administering a nucleotide sequence to a subject, comprisingadministering to a subject a duplexed parvovirus particle according tothe invention in a pharmaceutically acceptable carrier. Preferably, theduplexed parvovirus particle is administered in atherapeutically-effective amount to a subject in need thereof.

[0020] As a further aspect, the present invention provides a method ofdelivering a nucleotide sequence to a cell, comprising: contacting acell with a duplexed parvovirus particle comprising: a parvovirus capsidand a vector genome comprising: (i) a 5′ parvovirus terminal repeatsequence; (ii) a first heterologous nucleotide sequence; (iii) acentrally-located parvovirus terminal repeat sequence; (iv) a separateheterologous nucleotide sequence that is essentially completelycomplementary to the first heterologous nucleotide sequence (v) a 3′parvovirus terminal repeat sequence; wherein the duplexed vector genomeis capable under appropriate conditions of intrastrand base-pairingbetween the heterologous nucleotide sequences upon release from theparvovirus capsid.

[0021] According to this embodiment, preferably the parvovirus capsid isan AAV capsid, and the vector genome is approximately the size of thewild-type AAV genome. It is further preferred that the parvovirusterminal repeat sequences are AAV sequences. The cell may be contactedwith the duplexed parvovirus particle in vitro or in vivo.

[0022] These and other aspects of the present invention are described inmore detail in the description of the invention set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1. Virion DNA content of rAAV and duplexed vectors. Thedrawing illustrates the DNA content of the vectors used in this studyand the predicted conformation that they adopt upon release from thevirions. The transgenes expressed from the cytomegalovirus immediateearly promoter (CMV) are: green fluorescent protein (GFP), βgalactosidase (LacZ), mouse erythropoietin (mEpo). Neomycinphosphotransferase (neo) is expressed from the SV40 early promoter(SV40). The size, in nucleotides (nt) of each packaged DNA molecule isindicated. The self-complementary or duplexed (scAAV) GFP dimer and mEpovectors fold into a complete duplex DNA with one extra copy of theterminal repeat while the GFPneo, LacZ, and mEpoλ vectors requirecell-mediated DNA synthesis of the complementary strand.

[0024]FIG. 2. Vector fractionation on CsCl gradients. Virion DNA (vDNA)was extracted from CsCl gradient fractionated CMV-GFP (Panel a), GFPneo(Pan l b), and LacZ (Panel c) rAAV vectors. Alkaline agarose gels of thevDNA were Southern blotted and hybridized with a CMV-GFP DNA fragment.Markers at the left end of panel a were the excised vector sequencesfrom the plasmids used to generate the viral vectors (see results). Thenumber of unit length, ssDNA, vector copies per molecule are indicatedby 1×, 2×, and 4×. The viral vectors used in the experiments depicted inFIGS. 3 and 4 were from fractions a-11 or a-10 for CMV-GFP (as indicatedin the figure legends), fraction b-13 for GFPneo, and fraction c-12 forLacZ.

[0025]FIG. 3. Transduction efficiency of duplexed versus conventionalrAAV vectors in the absence and presence of co-infecting adenovirus. Theefficiencies of the three CsCl fractionated vectors (FIG. 1) werecompared in rapidly dividing HeLa cells infected with scAAV-GFP fraction11 (0.5 particles per cell), rAAV-GFPneo fraction 13 (2 particles percell), or rAAV-LacZ fraction 12 (0.5 particles per cell). Transductionwas quantified at 24 hours post-infection by counting GFP positive cellsusing fluorescence microscopy, or by fixing the cells and X-Gal.staining. The transducing efficiency was graphed as the number ofphysical particles per transducing unit, as determined by the number ofcells scoring positive for GFP or LacZ expression. Dark grey barsindicate transducing efficiency in the presence of Ad co-infection at 5pfu per cell.

[0026]FIG. 4. Transduction with duplexed and conventional rAAV vectorsin the presence of DNA synthesis inhibitor. (Panel a). HeLa cellcultures at 30% confluence were treated with the indicatedconcentrations of hydroxyurea 24 hr before infecting with 3.8×10⁶particles of the scAAV-GFP, ♦, (FIG. 2a, fraction 10), the homologousmonomer,  (FIG. 2, panel a, fraction 14), or rAAV-GFPneo, (FIG. 2,panel b, fraction 13). The HU treatment was maintained untiltransduction was assayed at 24 hr post-infection. Each data point wascalculated from the mean of the number of GFP positive cells in 10random fields independently of the total cell number, which was variabledue to the effect of hydroxyurea on cell division. (Panel b). The sameprocedure was used to evaluate transduction in the presence of theindicated concentrations of aphidicolin. Only the duplexed andhomologous monomer (fractions 10 and 14) were compared.

[0027]FIG. 5. In vivo transduction of mouse liver tissue with duplexedor single-stranded rAAV vectors. Ten week old Balb-c ByJ mice wereinfused with 2×10¹⁰ particles of either scAAV-CMV-mEpo, ♦, (n=4), orfull-length single-stranded rAAV-CMV-mEpoλ, ▴, (n=5), in 200 μl normalsaline by portal vein injection. One group of control mice was infusedwith normal saline, □, (n=4), and a single mouse, ◯, was phlebotomizedat the same 7-day intervals without prior surgery. Blood hematocrit wasused as a functional measure of mEpo expression.

[0028]FIG. 6 is a representation of a preferred template for producingthe duplexed parvovirus vectors of the invention.

[0029]FIG. 7 shows a CsCl density gradient of the rAAV-CMV-GFP Hpa-trsmutant vector.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will now be described with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown. This invention may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of theinvention to those skilled in the art.

[0031] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The terminology usedin the description of the invention herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. As used in the description of the inventionand the appended claims, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety.

[0032] Nucleotide sequences are presented herein by single strand only,in the 5′ to 3′ direction, from left to right, unless specificallyindicated otherwise. Nucleotides and amino acids are represented hereinin the manner recommended by the IUPAC-IUB Biochemical NomenclatureCommission, or (for amino acids) by either the one-letter code, or thethree letter code, both in accordance with 37 CFR §1.822 and establishedusage. See, e.g., Patentln User Manual, 99-102 (November 1990) (U.S.Patent and Trademark Office).

[0033] Except as otherwise indicated, standard methods known to thoseskilled in the art may be used for the construction of recombinantparvovirus and rAAV constructs, packaging vectors expressing theparvovirus rep and/or cap sequences, as well as transiently and stablytransfected packaging cells. Such techniques are known to those skilledin the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORYMANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al.CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates,Inc. and John Wiley & Sons, Inc., New York).

[0034] Parvoviruses are relatively small DNA animal viruses and containa linear, single-stranded DNA genome. The term “parvovirus” as usedherein encompasses the family Parvoviridae, includingautonomously-replicating parvoviruses and dependoviruses. The autonomousparvoviruses include members of the genera Parvovirus, Erythrovirus,Densovirus, Iteravirus, and Contravirus. Exemplary autonomousparvoviruses include, but are not limited to, mouse minute virus, bovineparvovirus, canine parvovirus, chicken parvovirus, feline panleukopeniavirus, feline parvovirus, goose parvovirus, and B19 virus. Otherautonomous parvoviruses are known to those skilled in the art. See,e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed.,Lippincott-Raven Publishers).

[0035] The genus Dependovirus contains the adeno-associated viruses(AAV), including but not limited to, AAV type 1, AAV type 2, AAV type 3,AAV type 4, AAV type 5, AAV type 6, avian AAV, bovine AAV, canine AAV,equine AAV, and ovine AAV. See, e.g., BERNARD N. FIELDS et al.,VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).

[0036] As used herein, the term “vector” or “gene delivery vector” mayrefer to a parvovirus (e.g., AAV) particle that functions as a genedelivery vehicle, and which comprises vDNA (i.e., the vector genome)packaged within a parvovirus (e.g., AAV) capsid. Alternatively, in somecontexts, the term “vector” may be used to refer to the vectorgenome/vDNA.

[0037] A “heterologous nucleotide sequence” will typically be a sequencethat is not naturally-occurring in the virus. Alternatively, aheterologous nucleotide sequence may refer to a viral sequence that isplaced into a non-naturally occurring environment (e.g., by associationwith a promoter with which it is not naturally associated in the virus).

[0038] As used herein, a “recombinant parvovirus vector genome” is aparvovirus genome (i.e., vDNA) into which a heterologous (e.g., foreign)nucleotide sequence (e.g., transgene) has been inserted. A “recombinantparvovirus particle” comprises a recombinant parvovirus vector genomepackaged within a parvovirus capsid.

[0039] Likewise, a “rAAV vector genome” is an AAV genome (i.e., vDNA)that comprises a heterologous nucleotide sequence. rAAV vectors requireonly the 145 base terminal repeats in cis to generate virus. All otherviral sequences are dispensable and may be supplied in trans (Muzyczka,(1992) Curr. Topics Microbiol. Immunol 158:97). Typically, the rAAVvector genome will only retain the minimal terminal repeat (TR)sequences so as to maximize the size of the transgene that can beefficiently packaged by the vector. A “rAAV particle” comprises a rAAVvector genome packaged within an AAV capsid.

[0040] The inventive parvovirus particles may be a “hybrid” particle inwhich the viral TRs and viral capsid are from different parvoviruses.Preferably, the viral TRs and capsid are from different serotypes of AAV(e.g., as described in international patent publication WO 00/28004,U.S. Provisional Application No. 60/248,920; and Chao et al., (2000)Molecular Therapy 2:619; the disclosures of which are incorporatedherein in their entireties). Likewise, the parvovirus may have a“chimeric” capsid (e.g., containing sequences from differentparvoviruses, preferably different AAV serotypes) or a “targeted” capsid(e.g., a directed tropism) as described in international patentpublication WO 00/28004.

[0041] Preferably, the inventive duplexed parvovirus particle has an AAVcapsid, which may further by a chimeric or targeted capsid, as describedabove.

[0042] The inventive “duplexed” parvovirus particles and vector genomesmay interchangeably be referred to herein as “dimeric” or“self-complementary” vectors. The duplexed parvovirus particles of theinvention comprise a parvovirus capsid containing a virion DNA (vDNA).The vDNA is self-complementary so that it may form a hairpin structureupon release from the viral capsid. The duplexed vDNA appears to provideto the host cell a double-stranded DNA that may be expressed (i.e.,transcribed and, optionally, translated) by the host cell without theneed for second-strand synthesis, as required with conventionalparvovirus vectors.

[0043] The duplexed parvovirus vector genome preferably containssufficient packaging sequences for encapsidation within the selectedparvovirus capsid (e.g, AAV capsid).

[0044] Those skilled in the art will appreciate that the duplexed vDNAmay not exist in a double-stranded form under all conditions, but hasthe ability to do so under conditions that favor annealing ofcomplementary nucleotide bases. Accordingly, the term “duplexedparvovirus vector” does not indicate that the vDNA is necessarily induplexed or double-stranded form (e.g., there is base-pairing betweenthe self-complementary strands) within the parvovirus capsid. Indeed,one skilled in the art will understand that the vDNA is likely not in adouble-stranded form while packaged within the parvovirus capsid.

[0045] Expression of a heterologous nucleotide sequence (as describedbelow) is preferably “enhanced” from the duplexed parvovirus vectors ofthe invention as compared with the comparable parvovirus (e.g., rAAV)vector. Preferably, gene expression may be detected from the duplexedparvovirus vector substantially more rapidly than from the comparablemonomeric parvovirus vector. For example, gene expression may bedetected in less than about 2 weeks, preferably less than about oneweek, more preferably less than about 72 hours, still more preferablyless than about 48 hours, and still more preferably less than about 24hours after administration of the duplexed parvovirus vector. Geneexpression may be detected by any method known in the art, e.g., bydetecting transcription, translation, or biological activity or aphenotypic effect resulting from expression of a heterologous nucleotidesequence (e.g., blood clotting time).

[0046] Alternatively, gene expression from the duplexed parvovirusvector may be “enhanced” in that higher levels of gene expression (asdefined in the preceding paragraph) are detected as compared with thecomparable monomeric parvovirus vector (e.g., rAAV vector). Comparisonsmay be made in the level of gene expression at the same time point afteradministration of virus. Alternatively, comparisons may be made betweenthe maximum level of gene expression achieved with each vector.

[0047] The duplexed parvovirus vectors of the invention mayadvantageously have improved transduction unit (tu) to particle ratiosas compared with conventional parvovirus vectors. Accordingly, thepresent invention also encompasses novel parvovirus vector compositionshaving an improved tu/particle ratio over compositions of conventionalparvovirus vectors (e.g., rAAV vectors). Preferably, the tu/particleratio is less than about 50:1, less than about 20:1, less than about15:1, less than about 10:1, less than about 8:1, less than about 7:1,less than about 6:1, less than about 5:1, less than about 4:1, or lower.There is no particular lower limit to the tu/particle ratio. Typically,the tu/particle ratio will be greater than about 1:1, 2:1, 3:1 or 4:1.

[0048] The term “template” or “substrate” is typically used herein torefer to a polynucleotide sequence that may be replicated to produce theduplexed parvovirus vDNA of the invention. For the purpose of vectorproduction, the template will typically be embedded within a largernucleotide sequence or construct, including but not limited to aplasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeastartificial chromosome (YAC) or a viral vector (e.g., adenovirus, herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral vectors, and thelike). Alternatively, the template may be stably incorporated into thechromosome of a packaging cell.

[0049] As used herein, the term “polypeptide” encompasses both peptidesand proteins, unless indicated otherwise.

[0050] As used herein, “transduction” or “infection” of a cell by AAVmeans that the AAV enters the cell to establish a latent or active(i.e., lytic) infection, respectively. See, e.g., BERNARD N. FIELDS etal., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-RavenPublishers). In embodiments of the invention in which a rAAV vector isintroduced into a cell for the purpose of delivering a nucleotidesequence to the cell, it is preferred that the AAV integrates into thegenome and establishes a latent infection.

[0051] Duplexed Parvovirus Vectors.

[0052] The present invention is based, in part, on the discovery that“duplexed” DNA parvovirus vectors (as described above) can beadvantageously employed for gene delivery. Furthermore, the presentinvestigations have demonstrated-that these duplexed parvovirus vectorsmay be more efficient than AAV vectors, e.g., improved transducing toparticle ratios, more rapid transgene expression, a higher level oftransgene expression, and/or more persistent transgene expression. Theinventors have further demonstrated that the duplexed parvovirus vectorsof the invention may be used for gene delivery to host cells that aretypically refractory to AAV transduction. Thus, these duplexedparvovirus vectors have a different (e.g., broader) host range than doAAV vectors.

[0053] The duplexed parvovirus vectors disclosed herein are dimericself-complementary (sc) polynucleotides (typically, DNA) packaged withina viral capsid, preferably a parvovirus capsid, more preferably, an AAVcapsid. In some respects, the viral genome that is packaged within thecapsid is essentially a “trapped” replication intermediate that cannotbe resolved to produce the plus and minus polarity parvovirus DNAstrands. Accordingly, the duplexed parvovirus vectors of the inventionappear to circumvent the need for host cell mediated synthesis ofcomplementary DNA inherent in conventional recombinant AAV (rAAV)vectors, thereby addressing one of the limitations of rAAV vectors.

[0054] This result is accomplished by allowing the virus to packageessentially dimeric inverted repeats of the single-stranded parvovirus(e.g., AAV) vector genome such that both strands, joined at one end, arecontained within a single infectious capsid. Upon release from thecapsid, the complementary sequences re-anneal to form transcriptionallyactive double-stranded DNA within the target cell.

[0055] The duplexed parvovirus vectors disclosed herein arefundamentally different from conventional parvovirus (e.g., rAAV)vectors, and from the parent parvovirus (e.g., AAV), in that the vDNAmay form a double-stranded hairpin structure due to intrastrand basepairing, and that both DNA strands are encapsidated. Thus, the duplexedparvovirus vector is functionally similar to double-stranded DNA virusvectors rather than the parvovirus from which it was derived. Thisfeature addresses a previously recognized shortcoming of rAAV mediatedgene transfer, which is the limited propensity of the desired targetcell to synthesize complementary DNA to the single-stranded genomenormally encapsidated by the Parvoviridae.

[0056] The viral capsid may be from any parvovirus, either an autonomousparvovirus or dependovirus, as described above. Preferably, the viralcapsid is an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6capsid). In general, the AAV1 capsid, AAV5 capsid, and AAV3 capsid arepreferred. The choice of parvovirus capsid may be based on a number ofconsiderations as known in the art, e.g., the target cell type, thedesired level of expression, the nature of the heterologous nucleotidesequence to be expressed, issues related to viral production, and thelike. For example, the AAV1 capsid may be advantageously employed forskeletal muscle, liver and cells of the central nervous system (e.g.,brain); AAV5 for cells in the airway and lung; AAV3 for bone marrowcells; and AAV4 for particular cells in the brain (e.g., appendablecells).

[0057] The parvovirus particle may be a “hybrid” particle in which theviral TRs and viral capsid are from different parvoviruses. Preferably,the viral TRs and capsid are from different serotypes of AAV (e.g., asdescribed in international patent publication WO 00/28004, U.S.provisional application No. 60/248,920; and Chao et al., (2000)Molecular Therapy 2:619; the disclosures of which are incorporatedherein in their entireties. Likewise, the parvovirus may have a“chimeric” capsid (e.g., containing sequences from differentparvoviruses) or a “targeted” capsid (e.g., a directed tropism) asdescribed in these publications.

[0058] As used herein, a “duplexed parvovirus particle” encompasseshybrid, chimeric and targeted virus particles. Preferably, the duplexedparvovirus particle has an AAV capsid, which may further by a chimericor targeted capsid, as described above.

[0059] A duplexed parvovirus vector according to the invention may beproduced by any suitable method. Preferably, the template for producingthe vDNA is one that preferentially gives rise to a duplexed, ratherthan monomeric vDNA (i.e., the majority of vDNA produced are duplexed)which has the capacity to form a double-stranded vDNA. Preferably, atleast about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of thereplication products from the template are duplexed.

[0060] In one particular embodiment, the template is a DNA moleculecomprising one or more terminal repeat (TR) sequences. The template alsocomprises a modified TR that cannot be resolved (i.e., nicked) by theparvovirus Rep proteins. During replication, the inability of Repprotein to resolve the modified TR will result in a stable intermediatewith the two “monomers” covalently attached by the non-resolvable TR.This “duplexed” molecule may be packaged within the parvovirus (AAV)capsid to produce a novel duplexed parvovirus vector.

[0061] While not wishing to be held to any particular theory of theinvention, it is likely that the virion genome is retained in asingle-stranded form while packaged within the viral capsid. Uponrelease from the capsid during viral infection, it appears that thedimeric molecule “snaps back” or anneals to form a double-strandedmolecule by intra-strand basepairing, with the non-resolvable TRsequence forming a covalently-closed hairpin structure at one end. Thisdouble-stranded vDNA obviates host cell mediated second-strandsynthesis, which has been postulated to be a rate-limiting step for AAVtransduction.

[0062] In preferred embodiments, the template further comprises aheterologous nucleotide sequence(s) (as described below) to be packagedfor delivery to a target cell. According to this particular embodiment,the heterologous nucleotide sequence is located between the viral TRs ateither end of the substrate. In further preferred embodiments, theparvovirus (e.g., AAV) cap genes and parvovirus (e.g., AAV) rep genesare deleted from the template (and the vDNA produced therefrom). Thisconfiguration maximizes the size of the heterologous nucleic acidsequence(s) that can be carried by the parvovirus capsid.

[0063] In one particular embodiment, the template for producing theinventive duplexed parvovirus vectors contains at least one TR at the 5′and 3′ ends, flanking a heterologous nucleotide sequence of interest (asdescribed below). The TR at one end of the substrate is non-resolvable,i.e., it cannot be resolved (nicked) by Rep protein. During replication,the inability of Rep protein to resolve one of the TRs will result in astable intermediate with the two “monomers” covalently attached by thenon-functional (i.e., non-resolvable) TR. The heterologous nucleotidesequence may be in either orientation with respect to the non-resolvableTR.

[0064] The term “flanked” is not intended to indicate that the sequencesare necessarily contiguous. For example, in the example in the previousparagraph, there may be intervening sequences between the heterologousnucleotide sequence and the TR. A sequence that is “flanked” by twoother elements, indicates that one element is located 5′ to the sequenceand the other is located 3′ to the sequence; however, there may beintervening sequences therebetween.

[0065] According to this embodiment, the template for producing theduplexed parvovirus vDNA of the invention is preferably about half ofthe size of the wild-type parvovirus genome (e.g., AAV) corresponding tothe capsid into which the vDNA will be packaged. Alternatively, stated,the template is preferably from about 40% to about 55% of wt, morepreferably from about 45% to about 52% of wt. Thus, the duplexed vDNAproduced from this template will preferably have a total size that isapproximately the size of the wild-type parvovirus genome (e.g., AAV)corresponding to the capsid into which the vDNA will be packaged, e.g.,from about 80% to about 105% of wt. In the case of AAV, it is well-knownin the art that the AAV capsid disfavors packaging of vDNA thatsubstantially deviate in size from the wt AAV genome. In the case of anAAV capsid, the template is preferably approximately 5.2 kb in size orless. In other embodiments, the template is preferably greater thanabout 3.6, 3.8, 4.0, 4.2, or 4.4 kb in length and/or less than about5.4, 5.2, 5.0 or 4.8 kb in length.

[0066] Alternatively stated, the heterologous nucleotide sequence(s)will typically be less than about 2.5 kb in length (more preferably lessthan about 2.4 kb, still more preferably less than about 2.2 kb inlength, yet more preferably less than about 2.1 kb in length) tofacilitate packaging of the duplexed template by the parvovirus (e.g.,AAV) capsid.

[0067] In another particular embodiment, the template itself isduplexed, i.e., is a dimeric self-complementary molecule. According tothis embodiment, the template comprises a resolvable TR at either end.The template further comprises a centrally-located non-resolvable TR (asdescribed above). In other words, each half of the template on eitherside of the non-resolvable TR is approximately the same length. Eachhalf of the template (i.e., between the resolvable and non-resolvableTR) comprises one or more heterologous nucleotide sequence(s) ofinterest. The heterologous nucleotide sequence(s) in each half of themolecule is flanked by a resolvable TR and the central non-resolvableTR.

[0068] The sequences in either half of the template are substantiallycomplementary (i.e., at least about 90%, 95%, 98%, 99% nucleotidesequence complementarity or more), so that the replication products fromthe template may form double-stranded molecules due to base-pairingbetween the complementary sequences. In other words, the template isessentially an inverted repeat with the two halves joined by thenon-resolvable TR.

[0069] Preferably, the heterologous nucleotide sequence(s) in each halfof the template are essentially completely self-complementary (i.e.,contains an insignificant number of mis-matched bases, or even nomismatched bases). It is also preferred that the two halves of thenucleotide sequence are essentially completely self-complementary.

[0070] According to this embodiment, the template (and the vDNA producedtherefrom) is preferably approximately the same size as the wt genomenaturally encapsulated by the parvovirus capsid (e.g., AAV), i.e., tofacilitate efficient packaging into the parvovirus capsid. For example,in the case of an AAV capsid, the template is preferably approximatelythe size of the wt AAV genome. In particular embodiments, the templateis approximately 5.2 kb in size or less. In other embodiments, thetemplate is preferably greater than about 3.6, 3.8, 4.0, 4.2, or 4.4 kbin length and/or less than about 5.4, 5.2, 5.0 or 4.8 kb in length. Asan alternative statement, the template is preferably in the range of 80%to 105% of the wildtype parvovirus genome (e.g., AAV).

[0071] The TR(s) (resolvable and non-resolvable) are preferably AAVsequences, with serotypes 1, 2, 3, 4, 5 and 6 being preferred. The term“terminal repeat” includes synthetic sequences that function as an AAVinverted terminal repeat, such as the “double-D sequence” as describedin U.S. Pat. No. 5,478,745 to Samulski et al., the disclosure of whichis incorporated in its entirety herein by reference. Resolvable AAV TRsaccording to the present invention need not have a wild-type TR sequence(e.g., a wild-type sequence may be altered by insertion, deletion,truncation or missense mutations), as long as the TR mediates thedesired functions, e.g., virus packaging, integration, and/or provirusrescue, and the like. Typically, but not necessarily, the TRs are fromthe same parvovirus, e.g., both TR sequences are from AAV2.

[0072] Those skilled in the art will appreciate that the viral Repprotein(s) used for producing the inventive duplexed vectors areselected with consideration for the source of the viral TRs. Forexample, the AAV5 TR interacts more efficiently with the AAV5 Repprotein.

[0073] The genomic sequences of the various autonomous parvoviruses andthe different serotypes of AAV, as well as the sequences of the TRs,capsid subunits, and Rep proteins are known in the art. Such sequencesmay be found in the literature or in public databases such as GenBank.See, e.g., GenBank Accession Numbers NC 002077, NC 001863, NC 001862, NC001829, NC 001729, NC 001701, NC 001510, NC 001401, AF063497, U89790,AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061,AH009962, AY028226, AY028223, NC 001358, NC 001540; the disclosures ofwhich are incorporated herein in their entirety. See also, e.g.,Chiorini et al., (1999) J. Virology 73:1309; Xiao et al., (1999) J.Virology 73:3994; Muramatsu et al., (1996) Virology 221:208;international patent publications WO 00/28061, WO 99/61601, WO 98/11244;U.S. Pat. No. 6,156,303; the disclosures of which are incorporatedherein in their entirety. An early description of the AAV1, AAV2 andAAV3 TR sequences is provided by Xiao, X., (1996), “Characterization ofAdeno-associated virus (AAV) DNA replication and integration,” Ph.D.Dissertation, University of Pittsburgh, Pittsburgh, Pa. (incorporatedherein it its entirety).

[0074] The non-resolvable TR may be produced by any method known in theart. For example, insertion into the TR will displace the nicking site(i.e., trs) and result in a non-resolvable TR. The designation of thevarious regions or elements within the TR are known in the art. Anillustration of the regions within the AAV TR is provided in FIG. 6 (seealso, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69, FIG. 5,3d ed., Lippincott-Raven Publishers). The insertion is preferably madeinto the sequence of the terminal resolution site (trs). Alternatively,the insertion may be made at a site between the Rep Binding Element(RBE) within the A element and the trs in the D element (see FIG. 6).The core sequence of the AAV trs site is known in the art and has beendescribed by Snyder et al., (1990) Cell 60:105; Snyder et al., (1993) J.Virology 67:6096; Brister & Muzyczka, (2000) J. Virology 74:7762;Brister & Muzyczka, (1999) J. Virology 73:9325 (the disclosures of whichare hereby incorporated by reference in their entireties). For example,Brister & Muzyczka, (1999) J. Virology 73:9325 describes a core trssequence of 3′-CCGGT/TG-5′ in the D element. Snyder et al., (1993) J.Virology 67:6096 identified the minimum trs sequence as 3′-GGT/TGA-5′,which substantially overlaps the sequence identified by Brister &Muzyczka.

[0075] Preferably, the insertion is in the region of the trs site. Theinsertion may be of any suitable length that will reduce orsubstantially eliminate (e.g., by 60%, 70%, 80%. 90%, 95% or greater)resolution of the TR. Preferably, the insertion is at least about 3, 4,5, 6, 10, 15, 20 or 30 nucleotides or more. There are no particularupper limits to the size of the inserted sequence, as long as suitablelevels of viral replication and packaging are achieved (e.g., theinsertion can be as long as 50, 100, 200 or 500 nucleotides or longer).

[0076] In another preferred embodiment, the TR may be renderednon-resolvable by deletion of the trs site. The deletions may extend 1,3, 5, 8, 10, 15, 20, 30 nucleotides or more beyond the trs site, as longas the template retains the desired functions. In addition to the trssite, some or all of the D element may be deleted. Deletions may furtherextend into the A element, however those skilled in the art willappreciate that it may be advantageous to retain the RBE in the Aelement, e.g., to facilitate efficient packaging. Deletions into the Aelement may be 2, 3, 4, 5, 8, 10, or 15 nucleotides in length or more,as long as the non-resolvable TR retains any other desired functions. Itis further preferred that some or all of the parvovirus (e.g., AAV)sequences going beyond the D element outside the TR sequence (e.g., tothe right of the D element in FIG. 6) be deleted to prevent geneconversion to correct the altered TR.

[0077] As still a further alternative, the sequence at the nicking sitemay be mutated so that resolution by Rep protein is reduced orsubstantially eliminated. For example, A and/or C bases may besubstituted for G and/or T bases at or near the nicking site. Theeffects of substitutions at the terminal resolution site on Rep cleavagehave been described by Brister & Muzyczka, (1999) J. Virology 73:9325(the disclosure of which is hereby incorporated by reference). As afurther alternative, nucleotide substitutions in the regions surroundingthe nicking site, which have been postulated to form a stem-loopstructure, may also be used to reduce Rep cleavage at the terminalresolution site (Id.).

[0078] Those skilled in the art will appreciate that the alterations inthe non-resolvable TR may be selected so as to maintain desiredfunctions, if any, of the altered TR (e.g., packaging, Rep recognition,site-specific integration, and the like).

[0079] In more preferred embodiments, the TR will be resistant to theprocess of gene conversion as described by Samulski et al., (1983) Cell33:135. Gene conversion at the non-resolvable TR will restore the trssite, which will generate a resolvable TR and result in an increase inthe frequency of monomeric replication products. Gene conversion resultsby homologous recombination between the resolvable TR and the alteredTR.

[0080] One strategy to reduce gene conversion is to produce virus usinga cell line (preferably, mammalian) that is defective for DNA repair, asknown in the art, as these cell lines will be impaired in their abilityto correct the mutations introduced into the viral template.

[0081] Alternatively, templates that have a substantially reduced rateof gene conversion can be generated by introducing a region ofnon-homology into the non-resolvable TR. Non-homology in the regionsurrounding the trs element between the non-resolvable TR and theunaltered TR on the template will reduce or even substantially eliminategene conversion.

[0082] Any suitable insertion or deletion may be introduced into thenon-resolvable TR to generate a region of non-homology, as long as geneconversion is reduced or substantially eliminated. Strategies thatemploy deletions to create non-homology are preferred. It is furtherpreferred that the deletion does not unduly impair replication andpackaging of the template. In the case of a deletion, the same deletionmay suffice to impair resolution of the trs site as well as to reducegene conversion.

[0083] As a further alternative, gene conversion may be reduced byinsertions into the non-resolvable TR or, alternatively, into the Aelement between the RBE and the trs site. The insertion is typically atleast about 3, 4, 5, 6, 10, 15, 20 or 30 nucleotides or more nucleotidesin length. There is no particular upper limit to the size of theinserted sequence, which may be as long as 50, 100, 200 or 500nucleotides or longer, however, it is preferred that the insertion doesnot unduly impair replication and packaging of the template.

[0084] In alternative embodiments, the non-resolvable TR may be anaturally-occurring TR (or altered form thereof) that is non-resolvableunder the conditions used. For example, the non-resolvable TR may not berecognized by the Rep proteins used to produce the vDNA from thetemplate. To illustrate, the non-resolvable TR may be an autonomousparvovirus sequence that is not recognized by AAV Rep proteins. As ananother illustrative example, the resolvable TR and Rep proteins may befrom one AAV serotype (e.g., AAV2), and the non-resolvable TR will befrom another AAV serotype (e.g., AAV5) that is not recognized by theAAV2 Rep proteins.

[0085] As a yet further alternative, the non-resolvable sequence may beany inverted repeat sequence that forms a hairpin structure and cannotbe cleaved by the Rep proteins.

[0086] As still a further alternative, a half-genome size template maybe used to produce a parvovirus particle carrying a duplexed vDNA,produced from a half-genome sized template, as described in the Examplesherein and by Hirata & Russell, (2000) J. Virology 74:4612. This reportdescribes packaging of paired monomers and transient RF intermediateswhen AAV genomes were reduced to less than half-size of the wtAAV genome(<2.5 kb). These investigators found that monomeric genomes were thepreferred substrate for gene correction by homologous recombination, andthat duplexed genomes functioned less well than did monomeric genomes inthis assay. This report did not investigate or suggest the use ofduplexed genomes as vectors for gene delivery.

[0087] Preferably, according to this embodiment, the template will beapproximately one-half of the size of the vDNA that can be packaged bythe parvovirus capsid. For example, for an AAV capsid, the template ispreferably approximately one-half of the wt AAV genome in length, asdescribed above.

[0088] The template (as described above) is replicated to produce aduplexed vector genome (vDNA) of the invention, which is capable offorming a double-stranded DNA under appropriate conditions. The duplexedmolecule is substantially self-complementary so as to be capable offorming a double-stranded viral DNA (i.e., at least 90%, 95%, 98%, 99%nucleotide sequence complementarity or more). Base-pairing betweenindividual nucleotide bases or polynucleotide sequences iswell-understood in the art. Preferably, the duplexed parvovirus viralDNA is essentially completely self-complementary (i.e., contains no oran insignificant number of mis-matched bases). In particular, it ispreferred that the heterologous nucleotide sequence(s) (e.g., thesequences to be transcribed by the cell) are essentially completelyself-complementary.

[0089] In general, the duplexed parvoviruses may containnon-complementarity to the extent that expression of the heterologousnucleotide sequence(s) from the duplexed parvovirus vector is moreefficient than from a corresponding monomeric vector.

[0090] The duplexed parvoviruses of the present invention provide thehost cell with a double-stranded molecule that addresses one of thedrawbacks of rAAV vectors, i.e., the need for the host cell to convertthe single-stranded rAAV vDNA into a double-stranded DNA. The presenceof any substantial regions of non-complementarity within the virion DNA,in particular, within the heterologous nucleotide sequence(s) willlikely be recognized by the host cell, and will result in DNA repairmechanisms being recruited to correct the mismatched bases, therebycounteracting the advantageous characteristics of the duplexedparvovirus vectors, e.g., the inventive vectors reduce or eliminate theneed for the host cell to process the viral template.

[0091] Production of Duplexed Parvovirus Vectors.

[0092] In general, methods of producing AAV vectors are applicable toproducing the duplexed parvovirus vectors of the invention; the primarydifference between the methods is the structure of the template orsubstrate to be packaged. To produce a duplexed parvovirus vectoraccording to the present invention, a template as described above willbe used to produce the encapsidated viral genome.

[0093] The template described above is preferably a DNA substrate andmay be provided in any form known in the art, including but not limitedto a plasmid, naked DNA vector, bacterial artificial chromosome (BAC),yeast artificial chromosome (YAC) or a viral vector (e.g., adenovirus,herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral vectors,and the like). Alternatively, the template may be stably incorporatedinto the genome of a packaging cell.

[0094] In one particular embodiment, the inventive parvovirus vectorsmay carry duplexed half-genome sized monomeric vDNA as described in theExamples herein. This means of providing cells with a duplexedparvovirus (e.g., AAV) virion DNA takes advantage of the rolling-hairpinmode of replication in which monomeric vDNA is generated from dimericinverted repeat intermediates (Cavalier-Smith et al., (1974) Nature250:467; Straus et al., (1976) Proc. Nat. Acad. Sci. USA 73:742). Whenthe genome is sufficiently small, the dimeric inverted repeatsthemselves can be encapsidated into the virion. This approach willgenerate a mixed population of monomeric and dimeric molecules. Theduplexed parvovirus vectors may be isolated by known techniques, e.g.,separation over a cesium chloride density gradient.

[0095] Duplexed parvovirus particles according to the invention may beproduced by any method known in the art, e.g., by introducing thetemplate to be replicated and packaged into a permissive or packagingcell, as those terms are understood in the art (e.g., a “permissive”cell can be infected or transduced by the virus; a “packaging” cell is astably transformed cell providing helper functions).

[0096] In one embodiment, a method is provided for producing a duplexedparvovirus particle, comprising: providing to a cell permissive forparvovirus replication (a) a nucleotide sequence encoding a template forproducing vector genome of the invention (as described in detail above);(b) nucleotide sequences sufficient for replication of the template toproduce a vector genome; (c) nucleotide sequences sufficient to packagethe vector genome into a parvovirus capsid, under conditions sufficientfor replication and packaging of the vector genome into the parvoviruscapsid, whereby duplexed parvovirus particles comprising the vectorgenome encapsidated within the parvovirus capsid are produced in thecell. Preferably, the parvovirus replication and/or capsid codingsequences are AAV sequences.

[0097] Any method of introducing the nucleotide sequence carrying thetemplate into a cellular host for replication and packaging may beemployed, including but not limited to, electroporation, calciumphosphate precipitation, microinjection, cationic or anionic liposomes,and liposomes in combination with a nuclear localization signal. Inembodiments wherein the template is provided by a virus vector, standardmethods for producing viral infection may be used.

[0098] Any suitable permissive or packaging cell known in the art may beemployed to produce the duplexed vectors. Mammalian cells are preferred.Also preferred are trans-complementing packaging cell lines that providefunctions deleted from a replication-defective helper virus, e.g., 293cells or other E1a trans-complementing cells. Also preferred aremammalian cells or cell lines that are defective for DNA repair as knownin the art, as these cell lines will be impaired in their ability tocorrect the mutations introduced into the viral template.

[0099] The template may contain some or all of the parvovirus (e.g.,AAV) cap and rep genes. Preferably, however, some or all of the cap andrep functions are provided in trans by introducing a packaging vector(s)encoding the capsid and/or Rep proteins into the cell. Most preferably,the template does not encode the capsid or Rep proteins. Alternatively,a packaging cell line is used that is stably transformed to express thecap and/or rep genes (see, e.g., Gao et al., (1998) Human Gene Therapy9:2353; Inoue et al., (1998) J. Virol. 72:7024; U.S. Pat. No. 5,837,484;WO 98/27207; U.S. Pat. No. 5,658,785; WO 96/17947).

[0100] In addition, helper virus functions are preferably provided forthe vector to propagate new virus particles. Both adenovirus and herpessimplex virus may serve as helper viruses for AAV. See, e.g., BERNARD N.FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-RavenPublishers). Exemplary helper viruses include, but are not limited to,Herpes simplex (HSV) varicella zoster, cytomegalovirus, and Epstein-Barrvirus. The multiplicity of infection (MOI) and the duration of theinfection will depend on the type of virus used and the packaging cellline employed. Any suitable helper vector may be employed. Preferably,the helper vector(s) is a plasmid, for example, as described by Xiao etal., (1998) J. Virology 72:2224. The vector can be introduced into thepackaging cell by any suitable method known in the art, as describedabove.

[0101] In one method, the inventive duplexed parvovirus vectors may beproduced by co-transfection of a rep/cap vector encoding AAV packagingfunctions and the template encoding the AAV vDNA into human cellsinfected with adenovirus (Samulski et al., (1989) J. Virology 63:3822).Under optimized conditions, this procedure can yield up to 10⁹infectious units of virus particles per ml. One drawback of this method,however, is that it results in the co-production of contaminatingwild-type adenovirus. Since several adenovirus proteins (e.g., fiber,hexon, etc.) are known to produce a cytotoxic T-lymphocyte (CTL) immuneresponse in humans (Yang and Wilson, (1995) J. Immunol. 155:2564; Yanget al., (1995) J. Virology 69:2004; Yang et al., (1994) Proc. Nat. Acad.Sci. USA 91:4407), this represents a significant drawback when usingthese rAAV preparations (Monahan et al., (1998) Gene Therapy 5:40).

[0102] Vector stocks free of contaminating helper virus may be obtainedby any method known in the art. For example, duplexed virus and helpervirus may be readily differentiated based on size. The duplexed virusmay also be separated away from helper virus based on affinity for aheparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973).Preferably, deleted replication-defective helper viruses are used sothat any contaminating helper virus is not replication competent. As afurther alternative, an adenovirus helper lacking late gene expressionmay be employed, as only adenovirus early gene expression is required tomediate packaging of the duplexed virus. Adenovirus mutants defectivefor late gene expression are known in the art (e.g., ts100K and ts149adenovirus mutants).

[0103] A preferred method for providing helper functions employs anon-infectious adenovirus miniplasmid that carries all of the helpergenes required for efficient AAV production (Ferrari et al., (1997)Nature Med. 3:1295; Xiao et al., (1998) J. Virology 72:2224).. The rAAVtiters obtained with adenovirus miniplasmids are forty-fold higher thanthose obtained with conventional methods of wild-type adenovirusinfection (Xiao et al., (1998) J. Virology 72:2224). This approachobviates the need to perform co-transfections with adenovirus (Holscheret al., (1994), J. Virology 68:7169; Clark et al., (1995) Hum. GeneTher. 6:1329; Trempe and Yang, (1993), in, Fifth Parvovirus Workshop,Crystal River, Fla.).

[0104] Other methods of producing rAAV stocks have been described,including but not limited to, methods that split the rep and cap genesonto separate expression cassettes to prevent the generation ofreplication-competent AAV (see, e.g., Allen et al., (1997) J. Virol.71:6816), methods employing packaging cell lines (see, e.g., Gao et al.,(1998) Human Gene Therapy 9:2353; Inoue et al., (1998) J. Virol.72:7024; U.S. Pat. No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785;WO 96/17947), and other helper virus free systems (see, e.g., U.S. Pat.No. 5,945,335 to Colosi).

[0105] Herpesvirus may also be used as a helper virus in AAV packagingmethods. Hybrid herpesviruses encoding the AAV Rep protein(s) mayadvantageously facilitate for more scalable AAV vector productionschemes. A hybrid herpes simples virus type I (HSV-1) vector expressingthe AAV-2 rep and cap genes has been described (Conway et al., (1999)Gene Therapy 6:986 and WO 00/17377, the disclosures of which areincorporated herein in their entireties).

[0106] In sum, the viral template to be replicated and packaged,parvovirus cap genes, appropriate parvovirus rep genes, and (preferably)helper functions are provided to a cell (e.g., a permissive or packagingcell) to produce parvovirus particles carrying the duplexed genome(i.e., the genome is capable of forming a “snap back” orself-complementary DNA after viral uncoating). The combined expressionof the rep and cap genes encoded by the template and/or the packagingvector(s) and/or the stably transformed packaging cell results in theproduction of a parvovirus particle in which a parvovirus capsidpackages a duplexed parvovirus genome according to the invention. Theduplexed parvovirus particles are allowed to assemble within the cell,and may then be recovered by any method known by those of skill in theart.

[0107] Alternatively, in vitro packaging approaches, as are known in theart, may also be used to produce the dimeric vDNA templates describedherein. To illustrate, the duplexed vDNA sequence may be amplified inbacteria using single-stranded M13 phage. The resolvable TRs at each endof the vDNA carried by the M13 will anneal to form a double-strandedsequence, which may be cleaved with a suitable restriction enzyme toexcise the dimeric vDNA from the M13 backbone. As yet a furtheralternative, PCR or other suitable amplification techniques may be usedto amplify the duplexed vDNA sequence from a dimeric self-complementarytemplate, as described above.

[0108] The reagents and methods disclosed herein may be employed toproduce high-titer stocks of the inventive parvovirus vectors,preferably at essentially wild-type titers. It is also preferred thatthe parvovirus stock has a titer of at least about 10⁵ transducing units(tu)/ml, more preferably at least about 10⁶ tu/ml, more preferably atleast about 10⁷ tu/ml, yet more preferably at least about 10⁸ tu/ml, yetmore preferably at least about 10⁹ tu/ml, still yet more preferably atleast about 10¹⁰ tu/ml, still more preferably at least about 10¹¹ tu/ml,or more.

[0109] Alternatively stated, the parvovirus stock preferably has a titerof at least about 1 tu/cell, more preferably at least about 5 tu/cell,still more preferably at least about 20 tu/cell, yet more preferably atleast about 50 tu/cell, still more preferably at least about 100tu/cell, more preferably still at least about 250 tu/cell, mostpreferably at least about 500 tu/cell, or even more.

[0110] Further, the duplexed parvovirus vectors of the invention, mayhave an improved transducing unit (tu)/particle ratio over conventionalparvovirus vectors. Preferably, the tu/particle ratio is less than about50:1, less than about 20:1, less than about 15:1, less than about 10:1,less than about 8:1, less than about 7:1, less than about 6:1, less thanabout 5:1, less than about 4:1, or lower. There is no particular lowerlimit to the tu/particle ratio. Typically, the tu/particle ratio will begreater than about 1:1, 2:1, 3:1 or 4:1.

[0111] Applications of the Present Invention.

[0112] A further aspect of the invention is a method of delivering anucleotide sequence to a cell using the duplexed parvovirus vectorsdescribed herein. The vector may be delivered to a cell in vitro or to asubject in vivo by any suitable method known in the art. Alternatively,the vector may be delivered to a cell ex vivo, and the cell administeredto a subject, as known in the art.

[0113] The present methods may be advantageously employed to providemore efficient transduction of target cells than wtAAV vectors. Toillustrate, the duplexed parvovirus vectors may transduce at a higherrate than wt AAV vectors. Alternatively, or additionally, the duplexedparvovirus vectors may provide for a more rapid onset of transgeneexpression, a higher level of transgene expression, and/or a longerpersistence of transgene expression than AAV vectors.

[0114] The inventive duplexed parvovirus vectors and methods may furtherfind use in methods of administering a nucleotide sequence to a cellthat is typically non-permissive for transduction by AAV, or is onlyinefficiently transduced by AAV. Exemplary cells include but are notlimited to dendritic cells, particular types of cancer or tumor cells,astrocytes, and bone marrow stem cells. Moreover, the methods disclosedherein may be advantageously practiced with non-replicating orslowly-replicating cells that only inefficiently support second-strandAAV synthesis, such as the liver, central nervous system (e.g., brain),and particular populations of cells within muscle (e.g., fast-twitchfibers).

[0115] Accordingly, the duplexed parvovirus vectors disclosed herein mayhave a distinct target cell range (e.g., a broader range of targetcells) as compared with rAAV vectors. While not wishing to be held toany particular theory of the invention, it appears that cells that arerefractory to transduction by rAAV may be permissive for the inventiveduplexed parvovirus vectors, which provide a double-stranded molecule tothe host cell. Thus, the present invention finds use for delivering anucleotide sequence to a cell that is non-permissive for conventionalrAAV-vectors or only poorly transduced by rAAV vectors because it cannotefficiently support second-strand synthesis of the viral DNA.

[0116] One of the characteristics of wtAAV vectors is the protracted lagperiod before high level transgene expression is observed. The duplexedparvovirus vectors disclosed herein may provide a more rapid andaggressive gene delivery system than wtAAV vectors because they obviatethe step of complementary strand synthesis.

[0117] Accordingly, the inventive duplexed parvovirus vectors find usein methods of treating cancer or tumors, e.g., by delivery ofanti-cancer agents or cancer antigens. In particular embodiments, theinventive methods are used to administer anti-cancer agents or cancerantigens to prevent metastasis, e.g., following surgical removal of aprimary tumor.

[0118] The inventive methods and duplexed parvovirus vectors may alsoadvantageously be used in the treatment of individuals with metabolicdisorders (e.g., omithine transcarbamylase deficiency). Such disorderstypically require a relatively rapid onset of expression of atherapeutic polypeptide by the gene delivery vector. As still a furtheralternative, the inventive vectors may be administered to provide agentsthat improve transplant survivability (e.g., superoxide dismutase) orcombat sepsis.

[0119] Moreover, the inventors have found that dendritic cells (DC),which are refractory to wtAAV vectors (Jooss et al., (1998) 72:4212),are permissive for the duplexed parvovirus vectors disclosed herein.Accordingly, as yet a further aspect, the present invention providesmethods of delivering a nucleotide sequence to DC, e.g., to induce animmune response to a polypeptide encoded by the nucleotide sequence.Preferably, the nucleotide sequence encodes an antigen from aninfectious agent or a cancer antigen.

[0120] As still a further aspect, the present invention may be employedto deliver a heterologous nucleotide sequence in situations in which itis desirable to regulate the level of transgene expression (e.g.,transgenes encoding hormones or growth factors, as described below). Themore rapid onset of transgene expression by the duplexed parvovirusvectors disclosed herein make these gene delivery vehicles more amenableto such treatment regimes than are rAAV vectors.

[0121] Any heterologous nucleotide sequence(s) (as defined above) may bedelivered according to the present invention. Nucleic acids of interestinclude nucleic acids encoding polypeptides, preferably therapeutic(e.g., for medical or veterinary uses) or immunogenic (e.g., forvaccines) polypeptides.

[0122] A “therapeutic polypeptide” is a polypeptide that may alleviateor reduce symptoms that result from an absence or defect in a protein ina cell or subject. Alternatively, a “therapeutic polypeptide” is onethat otherwise confers a benefit to a subject, e.g., anti-cancer effectsor improvement in transplant survivability.

[0123] Preferably, the heterologous nucleotide sequence or sequenceswill be less than about 2.5 kb in length (more preferably less thanabout 2.4 kb, still more preferably less than about 2.2 kb, yet morepreferably less than about 2.0 kb in length) to facilitate packaging ofthe duplexed template by the parvovirus (e.g., AAV) capsid. Exemplarynucleotide sequences encode Factor IX, Factor X, lysosomal enzymes(e.g., hexosaminidase A, associated with Tay-Sachs disease, or iduronatesulfatase, associated with Hunter Syndrome/MPS II), erythropoietin,angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase,tyrosine hydroxylase, as well as cytokines (e.g., α-interferon,β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin12, granulocyte-macrophage colony stimulating factor, lymphotoxin, andthe like), peptide growth factors and hormones (e.g., somatotropin,insulin, insulin-like growth factors 1 and 2, platelet derived growthfactor, epidermal growth factor, fibroblast growth factor, nerve growthfactor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor,glial derived growth factor, transforming growth factor-α and -β, andthe like), receptors (e.g., tumor necrosis factor receptor). In otherexemplary embodiments, the heterologous nucleotide sequence encodes amonoclonal antibodies, preferably a single-chained monoclonal antibodyor a monoclonal antibody directed against a cancer or tumor antigen(e.g., HER2/neu, and as described below). Other illustrativeheterologous nucleotide sequences encode suicide gene products(thymidine kinase, cytosine deaminase, diphtheria toxin, cytochromeP450, deoxycytidine kinase, and tumor necrosis factor), proteinsconferring resistance to a drug used in cancer therapy, and tumorsuppressor gene products.

[0124] As a further alternative, the heterologous nucleic acid sequencemay encode a reporter polypeptide (e.g., an enzyme such as GreenFluorescent Protein, alkaline phosphatase).

[0125] Alternatively, in particular embodiments of the invention, thenucleic acid of interest may encode an antisense nucleic acid, aribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs thateffect spliceosome-mediated trans-splicing (see, Puttaraju et al.,(1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No.6,083,702), interfering RNAs (RNAi) that mediate gene silencing (see,Sharp et al., (2000) Science 287:2431) or other non-translated RNAs,such as “guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like.

[0126] The parvovirus vector may also encode a heterologous nucleotidesequence that shares homology with and recombines with a locus on thehost chromosome. This approach may be utilized to correct a geneticdefect in the host cell.

[0127] The present invention may also be used to express an immunogenicpolypeptide in a subject, e.g., for vaccination. The nucleic acid mayencode any immunogen of interest known in the art including, but are notlimited to, immunogens from human immunodeficiency virus, influenzavirus, gag proteins, tumor antigens, cancer antigens, bacterialantigens, viral antigens, and the like.

[0128] The use of parvoviruses as vaccines is known in the art (see,e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S.Pat. Nos. 5,916,563 to Young et al., 5,905,040 to Mazzara et al., U.S.Pat. No. 5,882,652, U.S. Pat. No. 5,863,541 to Samulski et al.; thedisclosures of which are incorporated herein in their entirety byreference). The antigen may be presented in the parvovirus capsid.Alternatively, the antigen may be expressed from a heterologous nucleicacid introduced into a recombinant vector genome. Any immunogen ofinterest may be provided by the parvovirus vector. Immunogens ofinterest are well-known in the art and include, but are not limited to,immunogens from human immunodeficiency virus, influenza virus, gagproteins, tumor antigens, cancer antigens, bacterial antigens, viralantigens, and the like.

[0129] An immunogenic polypeptide, or immunogen, may be any polypeptidesuitable for protecting the subject against a disease, including but notlimited to microbial, bacterial, protozoal, parasitic, and viraldiseases. For example, the immunogen may be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoproteingene, or an equine influenza virus immunogen), or a lentivirus immunogen(e.g., an equine infectious anemia virus immunogen, a SimianImmunodeficiency Virus (SIV) immunogen, or a Human ImmunodeficiencyVirus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein,the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol andenv genes products). The immunogen may also be an arenavirus immunogen(e.g., Lassa fever virus immunogen, such as the Lassa fever virusnucleocapsid protein gene and the Lassa fever envelope glycoproteingene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia L1 orL8 genes), a flavivirus immunogen (e.g., a yellow fever virus immunogenor a Japanese encephalitis virus immunogen), a filovirus immunogen(e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such asNP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFSviruses), or a coronavirus immunogen (e.g., an infectious humancoronavirus immunogen, such as the human coronavirus envelopeglycoprotein gene, or a porcine transmissible gastroenteritis virusimmunogen, or an avian infectious bronchitis virus immunogen). Theimmunogen may further be a polio immunogen, herpes antigen (e.g., CMV,EBV, HSV immunogens) mumps immunogen, measles immunogen, rubellaimmunogen, diptheria toxin or other diptheria immunogen, pertussisantigen, hepatitis (e.g., hepatitis A or hepatitis B) immunogen, or anyother vaccine immunogen known in the art.

[0130] Alternatively, the immunogen may be any tumor or cancer cellantigen. Preferably, the tumor or cancer antigen is expressed on thesurface of the cancer cell. Exemplary cancer and tumor cell antigens aredescribed in S. A. Rosenberg, (1999) Immunity 10:281). Otherillustrative cancer and tumor antigens include, but are not limited to:BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2,BAGE, RAGE, NY-ESO-1, CDK4, β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE,SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994)Proc. Natl. Acad. Sci. USA 91:3515); Kawakami et al., (1994) J. Exp.Med., 180:347); Kawakami et al., (1994) Cancer Res. 54:3124), includingMART-1 (Coulie et al., (1991) J. Exp. Med. 180:35), gp100 (Wick et al.,(1988) J. Cutan. Pathol. 4:201) and MAGE antigen, MAGE-1, MAGE-2 andMAGE-3 (Van der Bruggen et al., (1991) Science, 254:1643); CEA, TRP-1,TRP-2, P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med.178:489); HER-2/neu gene product (U.S. Pat. No. 4,968,603), CA 125,LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50,SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 proteins, PSA,L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressorprotein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigens(international patent publication WO 90/05142); telomerases; nuclearmatrix proteins; prostatic acid phosphatase; papilloma virus antigens;and antigens associated with the following cancers: melanomas,metastases, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer,liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma,leukemias, uterine cancer, breast cancer, prostate cancer, ovariancancer, cervical cancer, bladder cancer, kidney cancer, pancreaticcancer and others (see, e.g., Rosenberg, (1996) Ann. Rev. Med.47:481-91).

[0131] Alternatively, the heterologous nucleotide sequence may encodeany polypeptide that is desirably produced in a cell in vitro, ex vivo,or in vivo. For example, the inventive vectors may be introduced intocultured cells and the expressed gene product isolated therefrom.

[0132] It will be understood by those skilled in the art that theheterologous nucleotide sequence(s) of interest may be operablyassociated with appropriate control sequences. For example, theheterologous nucleic acid may be operably associated with expressioncontrol elements, such as transcription/translation control signals,origins of replication, polyadenylation signals, and internal ribosomeentry sites (IRES), promoters, enhancers, and the like.

[0133] Those skilled in the art will appreciate that a variety ofpromoter/enhancer elements may be used depending on the level andtissue-specific expression desired. The promoter/enhancer may beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer may be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

[0134] Promoter/enhancer elements that are native to the target cell orsubject to be treated are most preferred. Also preferred arepromoters/enhancer elements that are native to the heterologous nucleicacid sequence. The promoter/enhancer element is chosen so that it willfunction in the target cell(s) of interest. Mammalian promoter/enhancerelements are also preferred. The promoter/enhance element may beconstitutive or inducible.

[0135] Inducible expression control elements are preferred in thoseapplications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery are preferablytissue-specific promoter/enhancer elements, and include muscle specific(including cardiac, skeletal and/or smooth muscle), neural tissuespecific (including brain-specific), liver specific, bone marrowspecific, pancreatic specific, spleen specific, retinal specific, andlung specific promoter/enhancer elements. Other induciblepromoter/enhancer elements include hormone-inducible and metal-inducibleelements. Exemplary inducible promoters/enhancer elements include, butare not limited to, a Tet on/off element, a RU486-inducible promoter, anecdysone-inducible promoter, a rapamycin-inducible promoter, and ametalothionein promoter.

[0136] In embodiments of the invention in which the heterologous nucleicacid sequence(s) will be transcribed and then translated in the targetcells, specific initiation signals are generally required for efficienttranslation of inserted protein coding sequences. These exogenoustranslational control sequences, which may include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

[0137] As a further advantage, the inventive duplexed parvovirus vectorsmay be distinguished from rAAV vectors in that the orientation of thecoding sequence with respect the resolvable TR is fixed and may becontrolled. Thus, for example, the orientation and expression of thetransgene may be controlled with respect to the putative transcriptionalcontrol elements within the resolvable TR. Moreover, control over theorientation of the transgene with respect to the non-resolvable TR mayprovide a greater level of control over the recombination productsbetween the genomes of co-infecting vectors. If either the closed end ofthe genome (i.e., near the non-resolvable TR) or the open end is apreferred substrate for intermolecular recombination, the orientation ofthe coding sequence within the recombination product can be predictedand controlled.

[0138] Finally, unlike rAAV vectors, the duplexed parvovirus vectors ofthe present invention are uniform in that they co-package both the plusand minus strands in a single molecule. This characteristic is desirablefrom the standpoint of producing a consistent clinical grade reagent.

[0139] Gene Transfer Technology.

[0140] The methods of the present invention also provide a means fordelivering heterologous nucleotide sequences into a broad range ofcells, including dividing and non-dividing cells. The present inventionmay be employed to deliver a nucleotide sequence of interest to a cellin vitro, e.g., to produce a polypeptide in vitro or for ex vivo genetherapy. The cells, pharmaceutical formulations, and methods of thepresent invention are additionally useful in a method of delivering anucleotide sequence to a subject in need thereof, e.g., to express animmunogenic or therapeutic polypeptide. In this manner, the polypeptidemay thus be produced in vivo in the subject. The subject may be in needof the polypeptide because the subject has a deficiency of thepolypeptide, or because the production of the polypeptide in the subjectmay impart some therapeutic effect, as a method of treatment orotherwise, and as explained further below.

[0141] In general, the present invention may be employed to deliver anyforeign nucleic acid with a biological effect to treat or ameliorate thesymptoms associated with any disorder related to gene expression.Illustrative disease states include, but are not-limited to: cysticfibrosis (and other diseases of the lung), hemophilia A, hemophilia B,thalassemia, anemia and other blood disorders, AIDs, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, epilepsy, and other neurological disorders, cancer, diabetesmellitus, muscular dystrophies (e.g., Duchenne, Becker), Gauchersdisease, Hurler's disease, adenosine deaminase deficiency, glycogenstorage diseases and other metabolic defects, retinal degenerativediseases (and other diseases of the eye), diseases of solid organs(e.g., brain, liver, kidney, heart), and the like.

[0142] Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, which may involve regulatory orstructural proteins, and which are typically inherited in a dominantmanner. For deficiency state diseases, gene transfer could be used tobring a normal gene into affected tissues for replacement therapy, aswell as to create animal models for the disease using antisensemutations. For unbalanced disease states, gene transfer could be used tocreate a disease state in a model system, which could then be used inefforts to counteract the disease state. Thus the methods of the presentinvention permit the treatment of genetic diseases. As used herein, adisease state is treated by partially or wholly remedying the deficiencyor imbalance that causes the disease or makes it more severe. The use ofsite-specific recombination of nucleic sequences to cause mutations orto correct defects is also possible.

[0143] The instant invention may also be employed to provide anantisense nucleic acid to a cell in vitro or in vivo. Expression of theantisense nucleic acid in the target cell diminishes expression of aparticular protein by the cell. Accordingly, antisense nucleic acids maybe administered to decrease expression of a particular protein in asubject in need thereof. Antisense nucleic acids may also beadministered to cells in vitro to regulate cell physiology, e.g., tooptimize cell or tissue culture systems.

[0144] Finally, the instant invention finds further use in diagnosticand screening methods, whereby a gene of interest is transiently orstably expressed in a cell culture system, or alternatively, atransgenic animal model.

[0145] In general, the present invention can be employed to deliver anyheterologous nucleic acid to a cell in vitro, ex vivo, or in vivo.

[0146] Subjects, Pharmaceutical Formulations, Vaccines, and Modes ofAdministration.

[0147] The present invention finds use in both veterinary and medicalapplications. Suitable subjects for ex vivo gene delivery methods asdescribed above include both avians and mammals, with mammals beingpreferred. The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys and pheasants. The term“mammal” as used herein includes, but is not limited to, humans,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.Human subjects are most preferred. Human subjects include neonates,infants, juveniles, and adults.

[0148] In particular embodiments, the present invention provides apharmaceutical composition comprising a virus particle of the inventionin a pharmaceutically-acceptable carrier and/or other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc. Forinjection, the carrier will typically be a liquid. For other methods ofadministration, the carrier may be either solid or liquid. Forinhalation administration, the carrier will be respirable, and willpreferably be in solid or liquid particulate form. As an injectionmedium, it is preferred to use water that contains the additives usualfor injection solutions, such as stabilizing agents, salts or saline,and/or buffers.

[0149] In general, a “physiologically acceptable carrier” is one that isnot toxic or unduly detrimental to cells. Exemplary physiologicallyacceptable carriers include sterile, pyrogen-free water and sterile,pyrogen-free, phosphate buffered saline. Physiologically-acceptablecarriers include pharmaceutically-acceptable carriers.

[0150] By “pharmaceutically acceptable” it is meant a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject without causing any undesirable biologicaleffects. Thus, such a pharmaceutical composition may be used, forexample, in transfection of a cell ex vivo or in administering a viralparticle or cell directly to a subject.

[0151] The parvovirus vectors of the invention maybe administered toelicit an immunogenic response (e.g., as a vaccine). Typically, vaccinesof the present invention comprise an immunogenic amount of infectiousvirus particles as disclosed herein in combination with apharmaceutically-acceptable carrier. An “immunogenic amount” is anamount of the infectious virus particles that is sufficient to evoke animmune response in the subject to which the pharmaceutical formulationis administered. Typically, an amount of about 10³ to about 10¹⁵ virusparticles, preferably about 10⁴ to about 10¹⁰, and more preferably about10⁴ to 10⁶ virus particles per dose is suitable, depending upon the ageand species of the subject being treated, and the immunogen againstwhich the immune response is desired. Subjects and immunogens are asdescribed above.

[0152] The present invention further provides a method of delivering anucleic acid to a cell. Typically, for in vitro methods, the virus maybe introduced into the cell by standard viral transduction methods, asare known in the art. Preferably, the virus particles are added to thecells at the appropriate multiplicity of infection according to standardtransduction methods appropriate for the particular target cells. Titersof virus to administer can vary, depending upon the target cell type andthe particular virus vector, and may be determined by those of skill inthe art without undue experimentation.

[0153] Recombinant virus vectors are preferably administered to the cellin a biologically-effective amount. A “biologically-effective” amount ofthe virus vector is an amount that is sufficient to result in infection(or transduction) and expression of the heterologous nucleic acidsequence in the cell. If the virus is administered to a cell in vivo(e.g., the virus is administered to a subject as described below), a“biologically-effective” amount of the virus vector is an amount that issufficient to result in transduction and expression of the heterologousnucleic acid sequence in a target cell.

[0154] The cell to be administered the inventive virus vector may be ofany type, including but not limited to neural cells (including cells ofthe peripheral and central nervous systems, in particular, brain cells),lung cells, retinal cells, epithelial cells (e.g., gut and respiratoryepithelial cells), muscle cells, dendritic cells, pancreatic cells(including islet cells), hepatic cells, myocardial cells, bone cells(e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells,keratinocytes, fibroblasts, endothelial cells, prostate cells, germcells, and the like. Alternatively, the cell may be any progenitor cell.As a further alternative, the cell can be a stem cell (e.g., neural stemcell, liver stem cell). As still a further alternative, the cell may bea cancer or tumor cell. Moreover, the cells can be from any species oforigin, as indicated above.

[0155] In particular embodiments of the invention, cells are removedfrom a subject, the parvovirus vector is introduced therein, and thecells are then replaced back into the subject. Methods of removing cellsfrom subject for treatment ex vivo, followed by introduction back intothe subject are known in the art (see, e.g., U.S. Pat. No. 5,399,346;the disclosure of which is incorporated herein in its entirety).Alternatively, the rAAV vector is introduced into cells from anothersubject, into cultured cells, or into cells from any other suitablesource, and the cells are administered to a subject in need thereof.

[0156] Suitable cells for ex vivo gene therapy are as described above.

[0157] The cells transduced with the inventive vector are preferablyadministered to the subject in a “therapeutically-effective amount” incombination with a pharmaceutical carrier. A “therapeutically-effective”amount as used herein is an amount that provides sufficient expressionof the heterologous nucleotide sequence delivered by the vector toprovide some improvement or benefit to the subject. Alternativelystated, a “therapeutically-effective” amount is an amount that willprovide some alleviation, mitigation, or decrease in at least oneclinical symptom in the subject. Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

[0158] In alternate embodiments, cells that have been transduced with avector according to the invention may be administered to elicit animmunogenic response against the delivered polypeptide. Typically, aquantity of cells expressing an immunogenic amount of the polypeptide incombination with a pharmaceutically-acceptable carrier is administered.An “immunogenic amount” is an amount of the expressed polypeptide thatis sufficient to evoke an active immune response in the subject to whichthe pharmaceutical formulation is administered. The degree of protectionconferred by the active immune response need not be complete orpermanent, as long as the benefits of administering the immunogenicpolypeptide outweigh any disadvantages thereof.

[0159] Dosages of the cells to administer to a subject will vary uponthe age, condition and species of the subject, the type of cell, thenucleic acid being expressed by the cell, the mode of administration,and the like. Typically, at least about 10² to about 10⁸, preferablyabout 10³ to about 10⁸ cells, will be administered per dose. Preferably,the cells will be administered in a “therapeutically-effective amount”.

[0160] A further aspect of the invention is a method of treatingsubjects in vivo with the inventive virus particles. Administration ofthe parvovirus particles of the present invention to a human subject oran animal in need thereof can be by any means known in the art foradministering virus vectors.

[0161] Exemplary modes of administration include oral, rectal,transmucosal, topical, transdermal, inhalation, parenteral (e.g.,intravenous, subcutaneous, intradermal, intramuscular, andintraarticular) administration, and the like, as well as direct tissueor organ injection, alternatively, intrathecal, direct intramuscular,intraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Alternatively, one may administer the virus in a local ratherthan systemic manner, for example, in a depot or sustained-releaseformulation.

[0162] The parvovirus vector administered to the subject may transduceany permissive cell or tissue. Suitable cells for transduction by theinventive parvovirus vectors are as described above.

[0163] In particularly preferred embodiments of the invention, thenucleotide sequence of interest is delivered to the liver of thesubject. Administration to the liver may be achieved by any method knownin the art, including, but not limited to intravenous administration,intraportal administration, intrabiliary administration, intra-arterialadministration, and direct injection into the liver parenchyma.

[0164] In other preferred embodiments, the inventive parvovirusparticles are administered intramuscularly, more preferably byintramuscular injection or by local administration (as defined above).Delivery to the brain is also preferred. In other preferred embodiments,the parvovirus particles of the present invention are administered tothe lungs.

[0165] The parvovirus vectors disclosed herein may be administered tothe lungs of a subject by any suitable means, but are preferablyadministered by administering an aerosol suspension of respirableparticles comprised of the inventive parvovirus vectors, which thesubject inhales. The respirable particles may be liquid or solid.Aerosols of liquid particles comprising the inventive parvovirus vectorsmay be produced by any suitable means, such as with a pressure-drivenaerosol nebulizer or an ultrasonic nebulizer, as is known to those ofskill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solidparticles comprising the inventive virus vectors may likewise beproduced with any solid particulate medicament aerosol generator, bytechniques known in the pharmaceutical art.

[0166] Dosages of the inventive parvovirus particles will depend uponthe mode of administration, the disease or condition to be treated, theindividual subject's condition, the particular virus vector, and thegene to be delivered, and can be determined in a routine manner.Exemplary doses for achieving therapeutic effects are virus titers of atleast about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10³, 10¹⁴, 10¹⁵transducing units or more, preferably about 10⁸-10¹³ transducing units,yet more preferably 10¹² transducing units.

[0167] In particular embodiments, the inventive parvovirus particles areadministered as part of a method of treating cancer or tumors byadministering anti-cancer agents (e.g., cytokines) or a cancer or tumorantigen. The parvovirus particle may be administered to a cell in vitroor to a subject in vivo or by using ex vivo methods, as described hereinand known in the art.

[0168] The term “cancer” has its understood meaning in the art, forexample, an uncontrolled growth of tissue that has the potential tospread to distant sites of the body (i.e., metastasize). Exemplarycancers include, but are not limited to, leukemias, lymriphomas, coloncancer, renal cancer, liver cancer, breast cancer, lung cancer, prostatecancer, ovarian cancer, melanoma, and the like. Preferred are methods oftreating and preventing tumor-forming cancers. The term “tumor” is alsounderstood in the art, for example, as an abnormal mass ofundifferentiated cells within a multicellular organism. Tumors can bemalignant or benign. Preferably, the inventive methods disclosed hereinare used to prevent and treat malignant tumors.

[0169] Cancer and tumor antigens according to the present invention havebeen described hereinabove. By the terms “treating cancer” or “treatmentof cancer”, it is intended that the severity of the cancer is reduced orthe cancer is at least partially eliminated. Preferably, these termsindicate that metastasis of the cancer is reduced or at least partiallyeliminated. It is further preferred that these terms indicate thatgrowth of metastatic nodules (e.g., after surgical removal of a primarytumor) is reduced or at least partially eliminated. By the terms“prevention of cancer” or “preventing cancer” it is intended that theinventive methods at least partially eliminate or reduce the incidenceor onset of cancer. Alternatively stated, the present methods slow,control, decrease the likelihood or probability, or delay the onset ofcancer in the subject.

[0170] Likewise, by the terms “treating tumors” or “treatment oftumors”, it is intended that the severity of the tumor is reduced or thetumor is at least partially eliminated. Preferably, these terms areintended to mean that metastasis of the tumor is reduced or at leastpartially eliminated. It is also preferred that these terms indicatethat growth of metastatic nodules (e.g., after surgical removal of aprimary tumor) is reduced or at least partially eliminated. By the terms“prevention of tumors” or “preventing tumors” it is intended that theinventive methods at least partially eliminate or reduce the incidenceor onset of tumors. Alternatively stated, the present methods slow,control, decrease the likelihood or probability, or delay the onset oftumors in the subject.

[0171] In other embodiments, cells may be removed from a subject withcancer or a tumor and contacted with the parvovirus particles of theinvention. The modified cell is then administered to the subject,whereby an immune response against the cancer or tumor antigen iselicited. This method is particularly advantageously employed withimmunocompromised subjects that cannot mount a sufficient immuneresponse in vivo (i.e., cannot produce enhancing antibodies insufficient quantities).

[0172] It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., α-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3,interleukin-4, interleukin5, interleukin6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, in particular embodiments of the invention,immunomodulatory cytokines (preferably, CTL inductive cytokines) areadministered to a subject in conjunction with the methods describedherein for producing an immune response or providing immunotherapy.

[0173] Cytokines may be administered by any method known in the art.Exogenous cytokines may be administered to the subject, oralternatively, a nucleotide sequence encoding a cytokine may bedelivered to the subject using a suitable vector, and the cytokineproduced in vivo.

[0174] Having now described the invention, the same will be illustratedwith reference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLE 1

[0175] Materials and Methods

[0176] Plasmids. The rAAV plasmids expressing green fluorescent protein(GFP) were constructed from the previously described pTR_(BS)UF-2 (agift from Nick Muzyczka). First, the humanized GFP coding sequence wasreplaced with the enhanced GFP (eGFP) (Clonetech) to create the plasmid,pTR-CMV-GFPneo. This plasmid generated the rAAV-GFPneo vector. Second,the Sal I fragment containing the neo coding region and SV40 promoterwas deleted to create pTR-CMV-GFP. The vector from this plasmid wasreferred to as rAAV-GFP in this report.

[0177] The plasmid, p43mEpo, a gift from Barry Byrne, contained themouse erythropoietin gene under the control of the CMV promoter andgenerated a rAAV replicon (rAAVmEpo) of less than half the wtAAV length.A longer version of this construct (pmEpo-λ) was made by inserting the2.3 kb Hind III fragment from λ phage into a Cla I site between thepolyadenylation signal and the downstream AAV terminal repeat. TherAAV-LacZ vector was generated from pDX11-LacZ, which has been describedelsewhere (McCown et al., (1996) Brain Research 713:99).

[0178] Viral vectors. Viral Vectors were generated in 293 cells (10⁸-10⁹cells per prep) by co-transfecting 3 plasmids containing: 1) thespecific rAAV construct, 2) the AAV rep and cap genes (pACG), or 3) theessential adenovirus helper genes (pXX-6; Xiao et al., (1998) J.Virology 72:2224). At 40 hr post-transfection, the cells were scrapedinto the media and lysed by three freeze-thaw cycles. The lysates wereincubated at 37° C. with 2 υg/ml DNase I until flocculent debris wasdispersed. The lysates were cleared by centrifugation and rAAV wasprecipitated using ammonium sulfate (Snyder et al., Production ofrecombinant adeno-associated virus vectors. In: Dracopoli et al.,editors. Current Protocols in Human Genetics. New York: John Wiley &Sons Ltd.: 1996. p. 12.1.1-12.2.23). The virus ppt. was resuspended with8 ml 10 mM Tris pH 8.0, 1 mM MgCl₂ and cesium chloride was added toreach a final density of 1.4 g/cm³ and a final volume 12.5 ml. Thesolution was centrifuged for 36 hrs at 38 krpm in an SW41 rotor.Fractions (0.75 ml) were collected by puncturing with a hypodermicneedle at the bottom of each tube and pumping the liquid to a fractioncollector. The vectors were stored at 4° C. in cesium chloride.

[0179] Virion DNA (vDNA) was extracted from 10 μl of each fraction bydigestion in 50 μl reactions containing 0.4 mg/ml protease K, 1%sarkosyl, and 10 mM EDTA at 50° C. for 1 hour, followed byphenol/chloroform extraction. The samples were diluted 3-fold with waterand precipitated with ethanol for analysis by alkaline agarose gelelectrophoresis and Southern blot hybridization.

[0180] Cells and infections. HeLa and HEK 293 cells were grown in DMEMmedia containing 10% FBS and Pen/Strep. Viral vector stocks were dilutedin media before adding to sub-confluent cultures and left on the cellsuntil GFP transduction was observed by fluorescence microscopy at 24hours post-infection.

[0181] For expression of erythropoietin in mouse livers, 200 μl normalsaline, containing 2×10¹⁰ physical particles of either conventional rAAVor duplexed virus (scAAV), was injected directly into the portal veinsof 10 week old Balb-c ByJ mice (Jackson Laboratory). Blood samples werecollected by retro-orbital phlebotomy at the time of infection and at7-day intervals for determination of hematocrit.

EXAMPLE 2

[0182] Generation of Duplexed Vectors

[0183] A rAAV plasmid construct (pTR-CMV-GFP), with a replicon size of2299 nucleotides, was used to generate a viral vector stock (rAAV-GFP)by conventional methods. The predicted size of the dimeric replicativeform of this vector was 4474 nucleotides (FIG. 1), which was 95.6% ofthe wt AAV genome length. The viral vectors were fractionated byisopycnic gradient centrifugation in CsCl and the vDNA content of eachfraction was analyzed on alkaline agarose gels (FIG. 2). Phospholmagerscans were used to quantify the vDNA specific bands from each fraction.Under denaturing conditions, the self-complementary dimer DNA (FIG. 2,panel a, fractions 10-13) ran at approximately twice the length of themonomeric genome. The hybridizing material in fractions 2-4 isunpackaged replicative form DNA that sediments at the bottom of thegradient. Although a DNase step was included in the vector purification(see methods), the treatment was not intended to be exhaustive and thismaterial proved to be DNase sensitive in subsequent experiments whilethe material in fractions 10-14 was DNase resistant (data not shown).Vectors containing mostly dimeric DNA genomes (fractions 10 and 11) weredesignated as duplexed or “self-complementary” virus (scAAV). Theinverted repeat structure of these molecules was confirmed byrestriction enzyme digestion (data not shown).

[0184] Two additional rAAV vectors (FIG. 1) were generated and purifiedin parallel, and analyzed in the same manner (FIG. 2, panels b and c).The first, rAAV-GFPneo, contained a neo gene in addition to the GFP andhad a replicating genome length of 3398 nucleotides. This was 72.6% ofthe wtAAV genome size, and was too large to be packaged as a dimer. Thesecond was a 4898 nucleotide rAAV-CMV-LacZ construct, which was slightlylarger (104.7%) than wtAAV genome size, but within the limit forefficient packaging (Dong et al., (1996) Human Gene Therapy 7:2101). Thelower density, higher mobility hybridizing material in fractions 14 and15 (FIG. 2, panel c) comprised genomes which had undergone deletions andthese fractions were not used in subsequent experiments.

EXAMPLE 3

[0185] Transduction with Duplexed versus Monomeric Vectors and Effectsof Ad co-Infection

[0186] The transducing efficiency of the scAAV-GFP (FIG. 2, panel a,fraction 11) was compared with the homologous monomer (fraction 13), aswell as the GFPneo and LacZ vectors (FIG. 2, panels b and c, fractions13 and 12, respectively) in HeLa cells infected at low multiplicity(FIG. 3). The particle numbers were calculated from the specific,full-length vDNA Phospholmager signals in each fraction on the Southernblot, after correction for monomeric versus dimeric DNA copy number.Thus, each duplexed virus contains two copies of the transgene as asingle molecule, in the inverted repeat orientation, while eachmonomeric particle contains one single-stranded copy.

[0187] The scAAV-GFP vector (fraction 11), containing approximately 90%dimer virus, yielded a 5.9:1 ratio of physical particles to transducingunits, thus bearing out the prediction of high transducing efficiency.Fraction 13 from the same gradient, conversely containing approximately80-90% monomer virus, had a 24.6:1 particle to transducing unit ratio.This 4-fold difference in efficiency represented a minimum differencewhen it was considered that the dimer contamination in the monomerfraction would have a greater impact on its transducing potential thanthe monomer component would contribute to the dimer fraction. Incontrast, the monomeric ssDNA GFPneo and LacZ vectors had particle totransducing unit ratios of 125:1 and 828:1, respectively, comparable topreviously reported efficiencies for these vectors (Fisher et al.,(1996) J. Virology 70:520; Zolotukhin et al., (1999) Gene Therapy6:973).

[0188] The transducing efficiency of conventional rAAV vectors can begreatly enhanced (up to 100-fold) by co-infection with Ad, or bytreatment with DNA damaging agents or other types of cell stress. Thisenhancement had been associated with the cell-mediated transformation ofthe ssDNA genome into active ds-DNA transcription templates. Because theduplexed vector contains the two complementary strands packaged as asingle molecule, it was predicted that transduction would be independentof enhancement by adenovirus. This was largely the case when HeLa cellswere co-infected with the duplexed vectors and 5 infectious units percell of adenovirus (FIG. 3). The number of GFP positive cells in theduplexed virus infected cultures was increased by only 1.6-fold, aneffect which could be attributed to the transcriptional effects ofadenovirus infection on the activity of the CMV promoter as previouslyreported (Clesham et al., (1998) Gene Therapy 5:174; Loser et al.,(1998) J. Virology 72:180). The monomer vector transduction rate wasincreased 2.4-fold by Ad co-infection, while the GFPneo and LacZ vectorswere induced 6.0-fold and 12.8-fold, respectively.

[0189] In sum, in cultured HeLa cells, the duplexed vector was greaterthan four-fold more efficient than the homologous vector containing onlya monomeric ss-DNA genome. This difference would likely be greater ifnot for the approximately 10-20% contamination of monomer fractions withdimer vectors. Consistent with this interpretation, the duplexed vectorwas 20-fold more efficient than a conventional rAAV-GFPneo vector and140-fold more efficient than a rAAV-LacZ vector.

EXAMPLE 4

[0190] Transduction with Duplexed Vectors in the Absence of Host CellDNA Synthesis

[0191] Because the vDNA of the duplexed vectors contained both DNAstrands on a single molecule, allowing efficient reannealing uponuncoating, it was predicted that these vectors would obviate the role ofhost-cell DNA synthesis in transduction. The scAAV-GFP vector wascompared with the homologous monomer, and the GFPneo vector, in HeLacells pretreated with hydroxyurea (HU) 24 hours before infection toinhibit host cell DNA synthesis. Hydroxyurea treatment was continued,uninterrupted, at the same concentrations following infection andmaintained on the cells for the following 24 hours, until GFPtransduction was scored.

[0192] Transduction from the scAAV-GFP was stimulated by up to 1.9 foldin response to increasing concentrations of HU (FIG. 4). Thisstimulation, similar in magnitude to that observed with Ad co-infection,was probably affected through a combination of transcriptionaltransactivation of the CMV promoter brought about by cell stress, andthe accumulation of GFP in the non-dividing cells. In contrast,transduction from the homologous monomer vector fraction was stimulatedat the lowest HU concentration and inhibited at higher concentrations.The residual transducing activity from the monomer vector at higher HUconcentrations, at a level approximately 5-fold lower than that of theduplexed virus fraction, is consistent with the 10-20% contamination ofthe monomer fractions with dimer containing particles (FIG. 2, panel a).The rAAV-GFPneo vector transduction was inhibited greater-than 10-foldunder the same conditions. Identical results were obtained by treatmentwith aphidicolin, a polymerase α/δ specific inhibitor (FIG. 4, panel b).This confirmed the hypothesis that duplexed vector transduction wasindependent of host-cell DNA synthesis.

EXAMPLE 5

[0193] Transduction by Duplexed Vectors in vivo

[0194] A different reporter was used for the comparison of duplexed andconventional single-stranded rAAV efficiency in vivo. Thedimer-producing construct contained only the mouse erythropoietin gene(mEpo) transcribed from the CMV promoter. The size of the replicatingelement of this minimal vector was 2248 nucleotides. The dimeric form ofthis molecule, 4372 nucleotides in length (FIG. 1), was 93% of the wtAAVgenome size, and was readily packaged. A second construct contained theidentical transgene, with the addition of a downstream heterologoussequence (λ phage) to bring the size of the recombinant vector to 4570nucleotides, or 98% of the wtAAV genome size. Previous studies have usedlambda phage DNA as a stuffer without deleterious effects on the vector(Muzyczka et al., (1992) Curr. Top. Microbiol. Immunol. 158:97). Bothvectors were purified by heparin-agarose chromatography. The smallervector was additionally purified on a CsCl gradient to isolate dimericDNA-containing virions (not shown). The two vector stocks werequantified using Southern blots from alkaline agarose gels to determinethe number of DNA-containing particles. In this case, approximately 25%of the particles in the dimer fraction contained two separate monomergenomes. Because they could not be separated from true dimer by density,and because their behavior has not been characterized, these werecounted as dimer particles, for the purpose of comparison to thefull-length vector, such that the dimer effect might only beunderestimated rather than overestimated.

[0195] Equal numbers of physical rAAV particles (2×10¹⁰ per animal in200 μl normal saline) were administered to mice by portal veininjection. The expression of the mEpo gene was evaluated by observingchanges in hematocrit at 7-day intervals. Control mice received eitherintraportal saline injections or were not operated, but phlebotomized at7-day intervals. Mice receiving the duplexed vector responded with arapid increase in hematocrit (FIG. 5), and with continuing increasesover the following two weeks. Considering the lag time betweenexpression of erythropoietin and the production of red blood cells, thissuggested that the duplexed vector was expressed at high levels withinthe first week. Mice which received the full-length, ssDNA vector didnot show a significant increase in hematocrit until 21 dayspost-injection, and did not reach levels comparable to the animalstreated with duplexed vector over the course of the experiment.

[0196] Infecting mice with scAAVmEpo leads to a faster response, and agreater rise in hematocrit, than the full-length ssDNA vector carryingthe same gene. These results support our observations in cultured cellsand is consistent with the view that the dimeric vectors are ready toexpress the transgene immediately upon uncoating and entry into thenucleus. The higher levels of expression ultimately achieved may reflectthe inability of many infects cells to form dsDNA from conventional rAAVand/or the loss/degradation of ssvDNA prior to the formation of duplex(Miao et al., (1998) Nature Genetics 19:13).

[0197] As we have demonstrated by pretreatment of cells with HU,transduction with the scAAV vector is independent of host cell DNAsynthesis. The ability to transduce cells in the absence of DNAsynthesis represents a fundamental departure in the biology of scAAVvectors from the parent virus, allowing them to function undercircumstances where conventional rAAV vectors would fail. Certain celltypes are extremely inefficient for rAAV transduction ostensibly due tothe inability to synthesize or recruit a complementary strand (Fisher etal., (1996) J. Virology 70:520; Alexander et al., (1996) Human GeneTherapy 7:841; Miao et al., (1998) Nature Genetics 19:13). The scAAVsuffers no such limitation and can be used with marker genes to directlydetermine whether a cell is permissive for rAAV transduction in allother steps irrespective of DNA synthesis.

[0198] Regardless of the ability of the target cell to make the rAAVcomplementary strand, it is clear that these reagents provide analternative AAV delivery system for genes that may require rapid onset.More importantly, our data suggest that scAAV vectors achieve overallhigher levels of therapeutic product when an identical number ofparticles is administered. Thus, scAAV vectors will prove useful where amore timely, robust, or quantitative response to vector dose isrequired. The potential for attaining critical levels of transgeneexpression at minimal dose is also important with respect to vectorproduction requirements for clinical trials and for minimizing patientexposure to virus.

EXAMPLE 6

[0199] Improved Substrates for Producing Duplexed Parvovirus Vectors

[0200] To streamline the production of duplexed vector stocks, and toeliminate the complications of mixed populations of duplex and monomergenomes, a mutant vector was created which generates only the dimergenomes (FIG. 6). This construct has a mutation in one TR, such that theRep nicking site (trs) is deleted, while the other TR is wild type. Theeffect is that rolling hairpin replication initiates from the wt end ofthe genome, proceeds through the mutant end without terminal resolution,and then continues back across the genome again to create the dimer. Theend product is a self-complementary genome with the mutant TR in themiddle and wt TRs now at each end. Replication and packaging of thismolecule then proceeds as normal from the wt TRs, except that thedimeric structure is maintained in each round.

[0201] Vector stocks of both rAAV-CMV-GFP-Hpa-trs andrAAV-CMV-mEpo-Hpa-trs have been generated using this mutant backgroundand analyzed the products on CsCl gradients as above (FIG. 7). Theseconstructs produce approximately 90% duplexed vectors. This will allowgreater yields of the duplexed parvovirus vector and the use ofiodixanol/heparin purification for these vectors without the additionalstep of CsCl density gradient purification.

[0202] The plasmid construct used to generate these vectors contained adeletion in the 5′ TR, relative to the coding strand of the expressedtransgene. This deletion includes all the D element and 3 bp of the Aelement, thus spanning the nicking site (FIG. 6). All AAV sequencesbetween the remainder of the A element and the transgene are deleted.This precludes homologous recombination between sequences flanking themutated TR and the wt TR, thus reducing the possibility of geneconversion as described by Samulski et al., (1983) Cell 33:135. Thisdeletion was constructed by cutting at unique restriction sitesimmediately 5′ to the transgene (Kpnl) and within the Amp gene of thebacterial plasmid sequences (Xmnl). The fragment removed, containing oneTR, was replaced with a fragment from a second rAAV plasmid, which hadbeen cut at the same site within the Amp gene, and at a synthetic Hpalsite previously inserted into the BalI site to the left of the A/Djunction.

[0203] In an alternative embodiment, a template for preferentiallyproducing duplexed vector is generated with a resolvable AAV TR at oneend and a modified AAV TR is produced by inserting a sequence into theTR. In one particular embodiment, the wt AAV plasmid psub201 is used toproduce this template (Samulski et al., (1987) J. Virology 61:3096).This construct contains a unique pair of Xba I sites as well as Pvullsites flanking the viral TRs. Two AAV plasmid intermediates derived frompsub201 , Hpa7 and Hpa9, have a unique Hpal linker (CCAATTGG) insertedat the Bal I site between nucleotide 121 and 122 (Hpa9) and between 4554and 4555 (Hpa7) in the TR sequence of the AAV genome, respectively(Xiao, X., (1996), “Characterization of Adeno-associated virus (AAV) DNAreplication and integration”, Ph.D. Dissertation, University ofPittsburgh, Pittsburgh, Pa.). Insertion of these linkers displaces thewt AAV nicking site inward away from the native position, resulting inan inability to be resolved by the AAV Rep protein after replication.

[0204] These substrates accumulate a dimeric intermediate until geneconversion takes place. Digestion of Hpa7 or Hpa9 with Hpal restrictionenzyme plus partial digestion with Xba I, results in novel TRs lackingthe wt AAV nicking site as well as the D element (from the left for Hpa9and from the right for Hpa7). This substrate is not suitable for geneconversion as described by Samulski et al., (1983) Cell 33:135, due tothe absence of the D element, and continues to accumulate a dimericreplication intermediate after viral infection. When starting with amolecule that is half-size or less of the wtAAV genome, thisintermediate is preferentially packaged by AAV capsids. These moleculesare dimeric in form (covalently linked through the modified TR), morespecifically, because they are self-complementary they provide a uniquesource of parvovirus vectors carrying double-stranded substrates. Thesevector particles bypass the rate-limiting step required for allcurrently utilized AAV vectors, namely, second-strand synthesis (seeFerrari et al., (1996) J. Virology 70:3227-34).

EXAMPLE 7

[0205] Transduction of Dendritic Cells

[0206] Dendritic cells (DC) are postulated to play important roles inantigen presentation and initiation of several T cell dependent immuneresponses. DC have been demonstrated to be more potentantigen-presenting cells (APC) than are macrophages or monocytes.Moreover, it has been reported that DC stimulate T cell proliferation upto ten-fold more efficiently than do monocytes (Guyre et al., (1997)Cancer Immunol. Immunother. 45:146, 147 col. 2). Accordingly, there arenumerous efforts to target vectors to dendritic cells so as to produce amore effective immune response. It has previously been reported that DCare refractory to AAV vectors (Jooss et al., (1998) J. Virology72:4212).

[0207] DC from two human patients were obtained and cultured in vitro.Cells from each patient were transduced with wtAAV-GFP vector orpHpa7GFP (duplexed vector, described in Example 1) at a MOI of 10. NoGFP expression was detected in cells transduced with wtAAV-GFP after 7days. In contrast, GFP expression was observed in 5-15% DC transducedwith dimeric pHpa7GFP vector.

[0208] These results suggest that the limiting step for wtAAVtransduction of DC is at level of host cell ability to mediatesecond-strand synthesis. The parvovirus vectors of the invention appearto obviate this step by providing the cell with a double-strandedsubstrate. Accordingly, the inventive dimeric parvovirus vectors have adifferent (e.g., broader) tropism and target cell range than do wtAAVvectors.

EXAMPLE 8

[0209] In vivo Administration of pHpa7GFP

[0210] To evaluate the tropism of the duplexed vectors in vivo, mice areadministered intramuscularly (im) with approximately 1.5×10¹¹ of thewtAAV-GFP or pHPA7GFP vectors described in Example 7. At various timespost-administration (e.g., 4, 8, 16, 32, 64 days, etc.), mice aresacrificed and autopsies performed to determine transgene expression invarious host cells and tissues. The onset, kinetics and persistence ofexpression are also evaluated and compared for the wtAAV anddouble-stranded vectors. Of particular interest are cells that aretypically refractory to wtAAV vectors such as bone marrow stem cells,astrocytes, and pulmonary epithelial cells. Also of interest arenon-replicating or slowly-replicating cells that inefficiently supportsecond-strand AAV synthesis such as muscle, liver and cells of thecentral nervous system.

[0211] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claimsand equivalents thereof.

1 1 1 175 DNA Artificial sequence Inverted terminal repeat from theAAV-2 vector plasmid pSub 201 1 ctacaaggaa cccctagtga tggagttggccactccctct ctgcgcgctc gctcgctcac 60 tgaggccgcc cgggcaaagc ccgggcgtcgggcgaccttt ggtcgcccgg cctcagtgag 120 cgagcgagcg cgcagagagg gagtggccaactccatcact aggggttcct tgtag 175

That which is claimed is:
 1. A duplexed parvovirus particle comprising: a parvovirus capsid a vector genome comprising in the 5′ to 3′ direction: (i) a 5′ parvovirus terminal repeat sequence; (ii) a first heterologous nucleotide sequence; (iii) a non-resolvable parvovirus terminal repeat sequence; (iv) a separate heterologous nucleotide sequence that is essentially completely complementary to said first heterologous nucleotide sequence; and (v) a 3′ parvovirus terminal repeat sequence; wherein said vector genome is capable under appropriate conditions of intrastrand base-pairing between the heterologous nucleotide sequences upon release from the parvovirus capsid.
 2. The duplexed parvovirus particle of claim 1, wherein said parvovirus terminal repeat sequences are adeno-associated virus (AAV) terminal repeat sequences.
 3. The duplexed parvovirus particle of claim 2, wherein said AAV terminal repeat sequences are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
 4. The duplexed parvovirus particle of claim 3, wherein said AAV terminal repeat sequences are AAV2 sequences.
 5. The duplexed parvovirus particle of claim 1, wherein the terminal resolution site (trs) is deleted from the non-resolvable terminal repeat sequence.
 6. The duplexed parvovirus particle of claim 5, wherein essentially all of the D element is deleted from said non-resolvable terminal repeat sequence.
 7. The duplexed parvovirus particle of claim 5, wherein the deletion in the D element of the non-resolvable terminal repeat sequence extends across the A/D junction into the A element.
 8. The nucleotide sequence of claim 1, wherein the non-resolvable parvovirus terminal repeat sequence comprises an insertion in the D element.
 9. The nucleotide sequence of claim 8, wherein the insertion in the D element is in the terminal resolution site (trs) sequence.
 10. The nucleotide sequence of claim 1, wherein the non-resolvable parvovirus terminal repeat sequence comprises one or more nucleotide substitutions in the terminal resolution site (trs) sequence.
 11. The duplexed parvovirus particle of claim 1, wherein the vector genome is approximately the size of the wild-type adeno-associated (AAV) genome.
 12. The duplexed parvovirus particle of claim 1, wherein a polypeptide is encoded by the vector genome.
 13. The duplexed parvovirus particle of claim 12, wherein the polypeptide is selected from the group consisting of endostatin, angiostatin, superoxide dismutase, erythropoietin, and a monoclonal antibody.
 14. The duplexed parvovirus particle of claim 1, wherein said parvovirus capsid is an adeno-associated virus (AAV) capsid.
 15. The duplexed parvovirus particle of claim 14, wherein said parvovirus capsid is selected from the group consisting of an AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6 capsid.
 16. The duplexed parvovirus particle of claim 15, wherein said parvovirus capsid is an AAV1 capsid.
 17. The duplexed parvovirus particle of claim 15, wherein said parvovirus capsid is an AAV2 capsid.
 18. The duplexed parvovirus particle of claim 1, wherein said parvovirus capsid is an adeno-associated virus (AAV) capsid and said parvovirus terminal repeat sequences are AAV terminal repeat sequences.
 19. The nucleotide sequence of claim 1, wherein the direction of transcription is toward the non-resolvable terminal repeat sequence.
 20. The nucleotide sequence of claim 1, wherein the direction of transcription is away from the non-resolvable terminal repeat sequence.
 21. The duplexed parvovirus particle of claim 1, wherein the 5′ and 3′ halves of the vector genome are essentially completely complementary to each other.
 22. A pharmaceutical formulation comprising a plurality of the duplexed parvovirus particles of claim 1 in a pharmaceutically acceptable carrier.
 23. A nucleotide sequence comprising a template for producing a virion DNA, the template comprising a heterologous nucleotide sequence flanked by a parvovirus terminal repeat sequence and a non-resolvable parvovirus terminal repeat sequence.
 24. The nucleotide sequence of claim 23, wherein said parvovirus terminal repeat sequences are adeno-associated virus (AAV) terminal repeat sequences.
 25. The nucleotide sequence of claim 24, wherein said AAV terminal repeat sequences are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
 26. The nucleotide sequence of claim 25, wherein said AAV terminal repeat sequences are AAV2 sequences.
 27. The nucleotide sequence of claim 23, wherein the terminal resolution site (trs) is deleted from the non-resolvable terminal repeat sequence.
 28. The nucleotide sequence of claim 27, wherein essentially all of the D element is deleted from said non-resolvable terminal repeat sequence.
 29. The nucleotide sequence of claim 27, wherein the deletion in the D element of the non-resolvable terminal repeat sequence extends across the A/D junction into the A element.
 30. The nucleotide sequence of claim 23, wherein the non-resolvable parvovirus terminal repeat sequence comprises an insertion in the D element.
 31. The nucleotide sequence of claim 30, wherein the insertion in the D element is in the terminal resolution site (trs) sequence.
 32. The nucleotide sequence of claim 23, wherein the non-resolvable parvovirus terminal repeat sequence comprises one or more nucleotide substitutions in the terminal resolution site (trs) sequence.
 33. The nucleotide sequence of claim 23, wherein the template is approximately one-half the size of the wild-type adeno-associated virus (AAV) genome.
 34. The nucleotide sequence of claim 23, wherein the heterologous nucleotide sequence or a complementary sequence thereto is a coding sequence for a polypeptide.
 35. The nucleotide sequence of claim 34, wherein the polypeptide is selected from the group consisting of endostatin, angiostatin, superoxide dismutase, erythropoietin, and a monoclonal antibody.
 36. The nucleotide sequence of claim 23, wherein the nucleotide sequence is selected from the group consisting of a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or a viral vector.
 37. The nucleotide sequence of claim 23, wherein the template is stably incorporated into the chromosome of a mammalian cell.
 38. A virion DNA produced from the nucleotide sequence of claim
 23. 39. A duplexed parvovirus particle comprising the virion DNA of claim
 38. 40. A nucleotide sequence comprising a dimeric template for producing a virion DNA, the template comprising in the 5′ to 3′ direction: a 5′ parvovirus terminal repeat sequence; a first heterologous nucleotide sequence; a non-resolvable parvovirus terminal repeat sequence; a separate heterologous nucleotide sequence that is essentially completely complementary to said first heterologous nucleotide sequence; and a 3′ parvovirus terminal repeat sequence; wherein virion DNA is capable under appropriate conditions of intrastrand base-pairing between the heterologous nucleotide sequences upon release from the parvovirus capsid.
 41. The nucleotide sequence of claim 40, wherein said parvovirus terminal repeat sequences are adeno-associated virus (AAV) terminal repeat sequences.
 42. The nucleotide sequence of claim 41, wherein said AAV terminal repeat sequences are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
 43. The nucleotide sequence of claim 42, wherein said AAV terminal repeat sequences are AAV2 sequences.
 44. The nucleotide sequence of claim 40, wherein the terminal resolution site (trs) is deleted from the non-resolvable terminal repeat sequence.
 45. The nucleotide sequence of claim 44, wherein essentially all of the D element is deleted from said non-resolvable terminal repeat sequence.
 46. The nucleotide sequence of claim 44, wherein the deletion in the D element of the non-resolvable terminal repeat sequence extends across the AND junction into the A element.
 47. The nucleotide sequence of claim 40, wherein the template is approximately the size of the wild-type adeno-associated (AAV) genome.
 48. The nucleotide sequence of claim 39, wherein the nucleotide sequence is selected from the group consisting of a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or a viral vector.
 49. The nucleotide sequence of claim 39, wherein the template is stably incorporated into the chromosome of a mammalian cell.
 50. The nucleotide sequence of claim 39, wherein the 5′ and 3′ halves of the dimeric template are essentially completely complementary to each other.
 51. A virion DNA produced from the nucleotide sequence of claim
 39. 52. A duplexed parvovirus particle comprising the virion DNA of claim
 51. 53. A cultured cell comprising the template of claim
 23. 54. A cultured cell comprising the template of claim
 39. 55. A method of producing a duplexed parvovirus particle, comprising providing to a cell permissive for parvovirus replication: (a) a nucleotide sequence encoding a template according to claim 23 or claim 39; (b) nucleotide sequences sufficient for replication of the template to produce a vector genome; (c) nucleotide sequences sufficient to package the vector genome into a parvovirus capsid, under conditions sufficient for replication and packaging of the vector genome into the parvovirus capsid, whereby duplexed parvovirus particles comprising the vector genome encapsidated within the parvovirus capsid are produced in the cell.
 56. The method of claim 55, further comprising the step of collecting the duplexed parvovirus particles.
 57. The method of claim 56, further comprising the step of lysing the cell prior to collecting the duplexed parvovirus particles.
 58. The method of claim 55, wherein the parvovirus rep and cap coding sequences are provided by a plasmid.
 59. The method of claim 55, wherein the parvovirus rep coding sequences are stably integrated into the cell.
 60. The method of claim 55, wherein the parvovirus cap coding sequences are stably integrated into the cell.
 61. The method of claim 55, wherein the nucleotide sequence comprising the template is a plasmid.
 62. The method of claim 55, wherein the template is stably integrated into the chromosome of a mammalian cell.
 63. A method of producing a duplexed parvovirus particle, comprising providing to a cell permissive for AAV replication: (a) a nucleotide sequence encoding a template according to claim 24 or claim 40; (b) AAV sequences sufficient for replication of the template to produce a vector genome; (c) AAV sequences sufficient to package the vector genome into an AAV capsid, under conditions sufficient for replication and packaging of the vector genome into the AAV capsid, whereby duplexed parvovirus particles comprising the parvovirus vector genome encapsidated within the AAV capsid are produced in the cell.
 64. The method of claim 63, further comprising providing helper virus sequences which provide the helper virus functions for a productive AAV infection, wherein the helper virus sequences cannot be packaged into infectious rAAV viral particles.
 65. The method of claim 63, further comprising the step of collecting the duplexed parvovirus particles.
 66. The method of claim 65, further comprising the step of lysing the cell prior to collecting the duplexed parvovirus particles.
 67. The method of claim 63, wherein the AAV rep and cap coding sequences are provided by a plasmid.
 68. The method of claim 67, wherein the plasmid further comprises helper virus sequences which provide the helper virus functions for a productive AAV infection.
 69. A method of delivering a nucleotide sequence to a cell, comprising contacting a cell with a duplexed parvovirus particle according to claim 1 under conditions sufficient for the duplexed parvovirus particle to enter the cell.
 70. The method of claim 69, wherein the cell is selected from the group consisting of a cancer cell, tumor cell, brain cell, muscle cell, airway epithelial cell, liver cell, dendritic cell, and eye cell.
 71. The method of claim 69, wherein a polypeptide is encoded by the vector genome.
 72. The method of claim 71, wherein the polypeptide is selected from the group consisting of endostatin, angiostatin, superoxide dismutase, erythropoietin, and a monoclonal antibody.
 73. The method of claim 69, wherein the parvovirus capsid is an adeno-associated virus (AAV) capsid.
 74. The method of claim 69, wherein the parvovirus terminal repeat sequences are adeno-associated virus (AAV) sequences.
 75. The method of claim 69, wherein the parvovirus capsid is an AAV capsid and the parvovirus terminal repeat sequences are AAV sequences.
 76. A method of administering a nucleic acid to a subject comprising administering the cell of claim 69 to a subject.
 77. A method of administering a nucleotide sequence to a subject, comprising administering to a subject a duplexed parvovirus particle according to claim 1 in a pharmaceutically acceptable carrier.
 78. The method of claim 77, wherein the parvovirus capsid is an adeno-associated virus (AAV) capsid.
 79. The method of claim 77, wherein the parvovirus terminal repeat sequences are adeno-associated virus (AAV) sequences.
 80. The method of claim 77, wherein the parvovirus capsid is an adeno-associated virus (AAV) capsid and the parvovirus terminal repeat sequences are AAV sequences.
 81. The method of claim 77, wherein the subject is selected from the group consisting of avian subjects and mammalian subjects.
 82. The method of claim 81, wherein the subject is a mammalian subject.
 83. The method of claim 82, wherein the subject is a human subject.
 84. The method of claim 82, wherein the subject is a cancer patient or tumor patient.
 85. The method of claim 82, wherein the duplexed parvovirus particle is administered by a route selected from the group consisting of oral, rectal, transmucosal, transdermal, inhalation, intravenous, subcutaneous, intradermal, intracranial, intramuscular, and intraarticular administration.
 86. The method of claim 82, wherein the duplexed parvovirus particle is administered to a site selected from the group consisting of a tumor, the brain, skeletal muscle, airway epithelium, the liver, and the eye.
 87. The method of claim 82, wherein a polypeptide is encoded by the vector genome.
 88. The method of claim 87, wherein the polypeptide is selected from the group consisting of selected from the group consisting of endostatin, angiostatin, superoxide dismutase, erythropoietin, and a monoclonal antibody. 